KR20080073725A - Uses of anti-cd40 antibodies - Google Patents

Abstract

Methods for treating a human patient for a cancer or pre-malignant condition that is associated with CD40-expressing cells are provided, where the human patient is heterozygous or homozygous for FcGRIIIa-158F (genotype V/F or F/F). Also provided are methods of inhibiting antibody production by B cells in a human patient who is heterozygous or homozygous for FcGRIIIa-158F (genotype V/F or F/F). The methods comprise administering to the human patient a therapeutically or prophylactically effective amount of an anti-CD40 antibody. Methods and kits for identifying a human patient with a cancer or pre-malignant condition that is treatable with an anti-CD40 antibody and which is refractory to treatment with rituximab (Rituxan(R)), as well as methods and kits for selecting an antibody therapy for treatment of a human patient having a cancer or pre-malignant condition that is refractory to treatment with rituximab (Rituxan(R)), are also provided. The methods of the present invention find use in treatment of cancers and pre-malignant conditions that are associated with CD40-expressing cells. These methods are particularly advantageous with respect to cancers and pre-malignant conditions that are associated with cells expressing both CD40 and CD20, as the methods enable the treatment of patients having a cancer or pre-malignant condition that is refractory to therapy with other oncotherapeutic agents such as anti-CD20 antibodies.

Description

Use of anti-CD40 antibody {USES OF ANTI 'CD40 ANTIBODIES}

The present invention relates in particular to the use of anti-CD40 antibodies in the treatment of cancer and precancerous conditions involving CD40-expressing cells.

Many members of the tumor necrosis factor (TNF) family of ligands and their corresponding receptors regulate growth of normal cells by inducing apoptosis or enhancing cell survival and proliferation. The balance between apoptosis and survival and proliferation signals maintains homeostasis of normal cells. At least 26 TNF family receptors and 18 TNF family ligands have been identified to date. Biologically active forms of both receptors and ligands are self-binding protein trimers. Dural and soluble forms of both receptors and ligands have been identified. Although the intracellular domains of the receptors do not share sequence homology, their extracellular domains include 40-amino acids, cysteine-rich repeats. Their cytoplasmic tail signals by interacting with two major groups of intracellular proteins: TNF receptor-associated factor (TRAF) and death domain (DD) -containing proteins. Interactions between at least six human TRAFs and TRAF-binding sites on the cytoplasmic tail of some of these receptors include AKT (referred to as serine / threonine kinase, protein kinase B or PKB), nuclear factor-κB (NF-κB), and mito Several signal pathways are disclosed, including gen-activated protein kinase (MAPK). See, eg, Younes and Kadin (2003) J CHn. Oncol. See 18: 3526-3534.

TNF family receptor member CD40 includes normal and neoplastic human B cells, dendritic cells, monocytes, macrophages, CD8 + T cells, endothelial cells, mononuclear and epithelial cells, and lung, breast, ovary, bladder, and colon cancer Is a 50-55 kDa cell-surface antigen present on the surface of many solid tumors. The CD40 ligand (CD40L) binds to the CD40 antigen on the B cell membrane, thereby stimulating B cell activation and proliferation to provide high levels of positive costimulatory signals that secrete B cells into plasma cells that secrete soluble immunoglobulins. CD40 activates TRAF-2, -3, -5, and -6, extracellular signal-regulating kinase (ERK), c-jun amino terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK), CD40L (either membrane-bound CD40L or soluble CD40L), including AKT, and NF-κB, will upregulate the various signaling pathways involved in the entry of CD40 (eg, Younes and Carbone (1999) Int. J. Biol.Markers 14: 135-143; van Kooten and Banchereau (2000) J. Leukoc. Biol. 67: 2-17).

Malignant B cells of the B-cell family tumor type express CD40 and appear to rely on CD40 signals for survival and proliferation. Transgenic cells of patients with low and high B-cell lymphoma, B-cell acute lymphoblastic leukemia, multiple myeloma, chronic lymphocytic leukemia, Waldenstrom's macroglobulinemia, and Hodgkin's disease express CD40. CD40 expression is also detected in acute myeloid leukemia and 50% of AIDS-associated lymphoma.

Many carcinomas and sarcomas also show high levels of CD40 expression, and the role of CD40 signaling in these cancer cells is not well known. CD40-expressing carcinomas include bladder carcinoma (Paulie et al. (1989) J. Immunol. 142: 590-595; Braesch-Andersen et al. (1989) J Immunol. 142: 562-567), breast carcinoma (Hirano et al. (1999) Blood 93: 2999-3007; Wingett et al. (1998) Breast Cancer Res. Treat. 50: 27-36); Prostate cancer (Rokhlin et al. (1997) Cancer Res. 57: 1758-1768), renal cell carcinoma (Kluth et al. (1997) Cancer Res. 57: 891-899), undifferentiated nasopharyngeal carcinoma (UNPC) (Agathanggelou) (1995) Am. J. Pathol. 147: 1152-1160), squamous cell carcinoma (SCC) (Amo et al. (2000) Eur. J. Dermatol. 10: 438-442; Posner et al. ( Clin. Cancer Res. 5: 2261-2270), papillary thyroid carcinoma (Smith et al. (1999) thyroid 9: 749-755), skin malignant melanoma (van den Oord et al. (1996) Am. J 149: 1953-1961), gastric carcinoma (Yamaguchi et al. (2003) Int. J. Oncol. 23 (6): 1697-702), and liver carcinoma (eg, Sugimoto et al. (1999). ) Hepatology 30 (4): 920-26, discussing human Hepatocyte carcinoma). For CD40-expressing sarcoma, see, eg, Lollini et al. ((1998) Clin. Cancer Res. 4 (8): 1843-849, studying human osteosarcoma and Ewing's sarcoma).

CD40 signaling protects immature B-cells and B-cell lymphomas from apoptosis induced from IgM or Fas (eg, Wang et al. (1995) J. Immunol. 155: 3722-3725). Outer membrane cell lymphoma cells express high levels of CD40 and the addition of exogenous CD40 ligands has been shown to enhance their survival and protect them from fludarabine-induced apoptosis (Clodi et al. (1998) Brit. J. Haematol. 103: 217-219). The role of CD40 signaling in malignant B cell survival and proliferation is to confer CD40 antigens on potential targets for anticancer therapy. Of course, antagonist anti-CD40 antibodies inhibit the proliferation and / or differentiation of malignant human B cells in vitro (eg, US Patent Application No. 20040109857). In addition, a murine (murine) model of invasive human lymphoma demonstrated the in vivo efficacy of anti-CD40 antibodies that promote animal survival. For example, Funakoshi et al. (1994) Blood 83: 2787-2794; Tutt et al. (1998) J Immunol. 161: 3176-3185; And Szocinski et al. (2002) Blood 100: 217-223.

CD40 ligand (CD40L), or CD40L, also known as CD154, is a 32-33 kDa transmembrane protein that exists in two small biologically active soluble forms, 18 kDa and 31 kDa, respectively (Graf et al. (1995) EMr. J. Immunol. 25: 1749-1754; Mazzei et al. (1995) J Biol. Chem. 270: 7025-7028; Pietravalle et al. (1996) J. Biol. Chem. 271: 5965-5967). CD40L is expressed as activated CD4 + T-helper cells rather than at rest (Lane et al. (1992) Eur. J. Immunol. 22: 2573-2578; Spriggs et al. (1992) J Exp. Med. 176: 1543-1550; and Roy et al. (1993) J Immunol. 151: 1-14). Both CD40 and CD40L were cloned and characterized (Stamenkovi et al. (1989) EMBO J. 8: 1403-1410; Armitage et al. (1992) Nature 357: 80-82; Lederman et al. (1992) J Exp Med. 175: 1091-1101; and Hollenbaugh et al. (1992) EMBO J. 11: 4313-4321). See also US Pat. No. 5,945,513, which describes human CD40L. Cells transfected with the CD40L gene, on their surface, express CD40L protein, can trigger B-cell proliferation and, in conjunction with other stimulatory signals, induce antibody production (Armitage et al. (1992) And US Patent No. 5,945,513). Patients with lymphoid malignancies, autoimmune diseases, cardiovascular disease, and essential thrombocytopenia have increased serum levels of soluble CD40L (sCD40L), which is not found in healthy subjects. Structural expression of CD40L includes mantle membrane-cell lymphoma, follicular lymphoma, marginal lymphoma, chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), and HIV-infected B-cell lymphoma Has been observed in a subset of patients with several B-cell lymphoid malignancies. For example, Clodi et al. (1998) Br. J. Haematol. 103: 270-275; Schattner et al. (1998) Blood 91: 2689-2697 '; Moses et al. (1997) Nat. Med. 3: 1242-1249; Trentin et al. (1997) Cancer Res. 57: 4940-4947; And Pham et al. (2002) Immunity 16: 37-50. CD40L plays an important role in cell contact-dependent interactions of CD40-expressing tumor B-cells in neoplastic vesicles or CD40-expressing Reed-Sternberg cells under the Hodgkin's disease region (Carbone et al. al. (1995) Am. J. Pathol. 147: 912-922). However, the mechanism of CD40L-mediated CD40 signaling that causes survival versus cell death due to malignant B-cells is not completely known. For example, in follicular lymphoma cells, downregulation of apoptosis-inducing TRAIL molecules (APO-2L) (Ribeiro et al. (1998) British J. Haematol. 103: 684-689) and excess of bcl-2 For expression, and for B-CLL, downregulation of CD95 (Fas / APO-1) (Laytragoon-Lewin et al. (1998) Eur. J. Haematol. 61: 266-271) has been proposed as a survival mechanism. In contrast, evidence in vesicle lymphoma suggests that CD40 activation is up-regulated by TNF (Worm et al. (1994) International Immunol. 6: 1883-1890) and CD95 molecules (Plumas et al. (1998) Blood 91: 2875-2885). Point out the fact that it induces regulation.

Human anti-CD40 monoclonal antibodies and many uses thereof are disclosed in WO 2005/044854, WO 2005/044304, WO 2005/044305, WO 2005/044306, WO 2005/044855, WO 2005/044307, and WO 2005/044294. Published patent applications. These applications specifically disclose human IgG ₁ anti-CD40 monoclonal antibodies designated CHIR-12.12 in the specification, which are generated by inoculation of transgenic mice containing human heavy chain locus and human κ light chain locus (Xeno mice ® technology; Abgenix, California).

As shown by FACS analysis, CHIR-12.12 specifically binds to human CD40 and can prevent CD40-ligand (CD40L) binding. CHIR-12.12 competed with CD40L pre-bound to cell surface CD40. CHIR-12.12 monoclonal antibodies are potent antagonists and inhibit in vitro CD40L-mediated proliferation of normal and malignant B cells. CHIR-12.12 monoclonal antibodies directly inhibit the survival and signaling pathways mediated by CD40L in normal human B-lymphocytes. In vitro, CHIR-12.12 kills primary cancer cells of NHL patients by ADCC. Dose-dependent anti-tumor activity is shown in xenograft human lymphoma models. CHIR-12.12 is currently in Phase I clinical trials for B-cell malignancies.

CD20 is a cell-surface antigen that is early expressed during B cell differentiation and remains on the cell surface through B cell development. CD20 is involved in B cell activation and is a clinically recognized therapeutic target, expressed at very high levels in neoplastic B cells (eg, Hooijberg et al. (1995) Cancer Research 55: 2627). Antibody targeting CD20, such as rituximab, Rituxan®, has been certified by the US Food and Drug Administration as a therapeutic for non-Hodgkin's lymphoma (eg, Boye et al. (2003) Ann. Oncol. 14: 520). Rituxan® has been shown to be effective in the treatment of low, moderate, and highly non-Hodgkin's lymphomas (NHL) and to be active against other B-cell malignancies (eg, Maloney et al. (1994) Blood 84: 2457-2466, McLaughlin et al. (1998) J Clin.Oncol 16: 2825-2833, Maloney et al (1997) Blood 90: 2188-2195, Hainsworth et al. (2000) Blood 95: 3052-3056, Colombat et (2001) Blood 97: 101-106, Coiffer et al. (1998) Blood 92: 1927-1932), Foran et al. (2000) J. Clin. Oncol. 18: 317-324, Anderson et al. (1997) Biochem. Soc. Trans. 25: 705-708, or Vose et al. (1999) Ann. Oncol. 10: 58a).

Although the exact mechanism of activity is unknown, evidence suggests that the anti-lymphoma effect of Rituxan® is partially dependent on complement-mediated cytotoxicity (CMC), antibody-dependent cell-mediated cytotoxicity (ADCC), inhibition of cell proliferation, and final It is pointed out that it is due to the direct induction of apoptosis. ADCC is the major mechanism of activity of many commercial and test monoclonal antibodies. Some patients, however, are resistant to treatment with Rituxan® (Witzig et al. (2002; J Clin. Oncol. 20: 3262, Grillo-Lopez et al. (1998) J Clin. Oncol. 16: 2825, Or Jazirehi et al. (2003) MoI. Cancer Ther. 2: 1183-1193.For example, some patients lose CD20 expression on malignant B cells after anti-CD20 antibody therapy (Davis et al. (1999). (Clin. Cancer Res. 5: 611) .On closer examination, 30% to 50% of low-grade NHL patients do not show a clinical response to this monoclonal antibody (Hainsworth et al. (2000) Blood 95: 3052-3056). Colombat et al. (2001) Blood 97: 101-106) The clinical activity of rituximab on NHL has been shown to correlate with the FcγRIIIa genotype of patients FcγRIIIa 158aa of V / V or V / F. Patients with polymorphism are more sensitive to rituximab than patients with F / F (eg, Cartron et al. (2002) Blood 99 (3): 754-758 or Dall'Ozzo et al. Cancer Res). (2004) 64: 4664-466 9) In patients with developing resistance to this monoclonal antibody or with B-cell lymphoma resistant to initial therapy of this antibody, an alternative form of therapeutic intervention is needed.

Thus, there is a continuing demand for new therapeutic agents and new therapeutic strategies for cancer and precancerous conditions. In particular, there is a need for new therapeutic strategies in patients who are homozygous or heterozygous for FcγRIIIa-158F and are resistant to treatment with anti-CD20 antibodies such as Rituximab (Rituxan®) and the like. Moreover, antibodies capable of killing malignant cells that do not require conjugates will be drugs that can be manufactured at low cost, with fewer side effects.

Summary of the Invention

The present invention provides a method for treating a cancer or precancerous condition associated with CD40-expressing cells of a human patient, wherein the human patient is heterozygous or homozygous for FcγRIIIa-158F (genotype V / F or F / F) to be. The method comprises administering a therapeutically or prophylactically effective amount of an anti-CD40 antibody to a human patient. The present invention relates to the preparation of an anti-CD40 antibody in the manufacture of a medicament for the treatment of cancer or precancerous conditions involving CD40-expressing cells in human patients who are heterozygous or homozygous for FcγRIIIa-158F (genotype V / F or F / F). Provides a therapeutically or prophylactically effective amount of use.

The invention also relates to antibodies by B cells in human patients that are heterozygous or homozygous (genotype V / F or F / F) to FcγRIIIa-158F, comprising administering an effective amount of an anti-CD40 antibody to a human patient. It provides a way to suppress production. The present invention provides the use of an effective amount of an anti-CD40 antibody in the manufacture of a medicament for the inhibition of antibody production by B cells in human patients who are heterozygous or homozygous for FcγRIIIa-158F (V / F or F / F). .

The present invention also provides methods and kits for identifying human patients treatable with anti-CD40 antibodies and who have cancer or precancerous conditions resistant to the treatment of Rituximab (Rituxan®). In one embodiment of the invention, the method comprises: a) identifying a human patient with a cancer or precancerous condition associated with CD40-expressing cells resistant to the treatment of Rituximab; And b) determining the FcγRIIIa-158 genotype (V / V, V / F or F / F) of the human patient; Here, if the human patient is heterozygous or homozygous for FcγRIIIa-158F (genotype V / F or F / F), the cancer or precancerous condition can be treated with anti-CD40 antibody. The present invention may further comprise administering a therapeutically or prophylactically effective amount of an anti-CD40 antibody to a human patient identified using this method. The kits of the invention provided for the identification of human patients with cancer or precancerous conditions treatable with an anti-CD40 antibody include reagents for determining the FcγRIIIa-158 genotype of human patients.

The invention also provides methods and kits for selecting therapeutic antibody therapies for human patients with cancer or precancerous conditions resistant to the treatment of Rituximab (Rituxan®). In one embodiment of the invention, the method comprises: a) identifying a human patient with a cancer or precancerous condition associated with CD40-expressing cells that is resistant to the treatment of Rituximab; And b) determining the FcγRIIIa-158 genotype (V / V, V / F or F / F) of the human patient; Wherein if the human patient is heterozygous or homozygous for FcγRIIIa-158F (genotype V / F or F / F), the anti-CD40 antibody is selected for treatment of cancer or precancerous conditions. The invention may further comprise administering a therapeutically or prophylactically effective amount of an anti-CD40 antibody to a human patient identified using this method. The kits of the invention provided for selecting therapeutic antibody therapies for human patients with cancer or precancerous conditions associated with anti-CD40 expressing cells include reagents for determining the FcγRIIIa-158 genotype of human patients.

The invention also provides a method for treating a cancer or precancerous condition associated with CD40-expressing cells of a human patient, the method comprising administering a slow-internalizing antibody to the human patient. In one embodiment of the invention, when a therapeutically or prophylactically effective amount of an anti-CD40 antibody is administered to a human patient, the anti-CD40 antibody is not significantly internalized by the CD40-expressing cells after their administration. In another embodiment of the invention, if a therapeutically or prophylactically effective amount of an anti-CD40 antibody is administered to a human patient, the anti-CD40 antibody is substantially uniformly distributed on the surface of the CD40-expressing cells after their administration. Will remain. In another embodiment of the invention, when anti-CD40 antibodies are administered to a human patient, a therapeutically or prophylactically effective amount of the anti-CD40 antibodies will be present on the surface of CD40-expressing cells in the human patient after their administration.

Anti-CD40 antibodies used according to the invention specifically bind to CD40 antigens. In one embodiment of the invention, the anti-CD40 antibody, particularly monoclonal antibody, used in the methods of the invention, has a strong binding affinity for human FcγRIIIa-158V, a strong binding affinity for human FcγRIIIa-158F, or human FcγRIIIa Strong binding affinity for -158V and FcγRIIIa-158F. As part of this embodiment of the invention, the anti-CD40 antibody is a two FcγRIIIa amino acid 158 allotype for natural killer (NK) cells of human patients with binding properties suitable for inducing potential antibody-dependent cytotoxicity (ADCC) It can couple to either (V or F). Suitable anti-CD40 antibodies include, but are not limited to, anti-CD40 antibodies without significant agonist activity, including, for example, anti-CD40 antibodies that are antagonists of CD40-CD40L signal against CD40-expressing cells. In one embodiment of the invention, the anti-CD40 antibody comprises: a) monoclonal antibody CHIR-12.12; b) monoclonal antibodies produced by hybridoma cell line 12.12; c) a sequence represented by SEQ ID NO: 2, a sequence represented by SEQ ID NO: 4, a sequence represented by SEQ ID NO: 5, both sequences represented by SEQ ID NO: 2 and SEQ ID NO: 4, and SEQ ID NO Monoclonal antibodies comprising an amino acid sequence selected from the group consisting of both sequences represented by: 2 and SEQ ID NO: 5; d) a nucleic acid comprising a nucleotide sequence selected from the group consisting of a sequence represented by SEQ ID NO: 1, a sequence shown in SEQ ID NO: 3, and both sequences shown in SEQ ID NO: 1 and SEQ ID NO: 3 Monoclonal antibodies having an amino acid sequence encoded by a molecule; e) monoclonal antibodies that bind to epitopes bindable to monoclonal antibodies produced by the hybridoma cell line 12.12; f) a monoclonal antibody that binds to an epitope comprising residues 82-87 of the human CD40 sequence represented by SEQ ID NO: 7 or SEQ ID NO: 9; g) a monoclonal antibody that binds to an epitope comprising residues 82-89 of the human CD40 sequence represented by SEQ ID NO: 7 or SEQ ID NO: 9; h) monoclonal antibodies that compete with monoclonal antibody CHIR-12.12 in a competitive binding assay; i) the monoclonal antibody of item a) or the monoclonal antibody of any one of item c) -h) above, wherein the antibody is a recombinantly produced monoclonal antibody; And j) a monoclonal antibody which is an antigen-binding fragment of the monoclonal antibody of any one of items a) -i) above, wherein the fragment has the ability to specifically bind a human CD40 antigen. Is selected from.

The methods of the present invention find use in the treatment of cancer and precancerous conditions associated with CD40-expressing cells. Examples include, but are not limited to, B-cell family cancer, non-B cell hematologic malignancies such as acute myeloid leukemia, solid tumors, and cancer or precancerous conditions associated with CD20-expressing cells. . The methods of the invention are particularly advantageous for cancer and precancerous conditions associated with cells expressing both CD40 and CD20. In this manner, the present invention is cancer or precancer resistant to therapy by other tumor therapeutics, including anti-CD20 antibodies in patients who are homozygous or heterozygous for FcγRIIIa-158F (genotype V / F or F / F). Enable treatment of patients with conditions.

1A-1F show the results of analysis of antibody-dependent cellular cytotoxicity (ADCC) of six cell lines.

2A-2D show the results of analysis of antibody-dependent cellular cytotoxicity (ADCC) in CLL patient cells (n = 8).

3 summarizes the results of analysis of ADCC in CLL patient cells (n = 9).

4 shows the results of analysis of ADCC in CLL patient cells using NK effector cells from two different donors.

5 shows quantitative results of CD40 and CD20 cell-surface expression in CLL patient cells and normal B cells.

6 summarizes ADCC activity of cells by quantified CD40 and CD20 cell-surface expression.

FIG. 7 is a bar graph showing the level of cell-surface binding CHIR-12.12 for Daudi and ARH77 cell lines.

8 shows the results of internalization experiments of CHIR-12.12 and rituximab in CLL patient cells by FACS analysis.

9 shows the results of internalization experiments of CHIR-12.12 and rituximab in normal B cells by confocal microscopy of FITC-labeled antibodies.

10 shows the results of internalization experiments of CHIR-12.12 and rituximab in CLL patient cells by confocal microscopy of Alexa488-labeled antibody.

11 summarizes the relationship between ADCC activity and internalization.

12 is a bar graph showing the maximum percentage of specific lysis of Daudi cells by CHIR-12.12 or Rituximab using purified NK effector cells of donors with other FcγRIIIa genotypes.

FIG. 13 is a bar graph showing ADCC titer (ED ₅₀ ) of CHIR-12.12 or rituximab on Daudi cells in purified NK effector cells of donors with other FcγRIIIa genotypes.

FIG. 14 summarizes the competitive ADCC of CHIR-12.12 or Rituximab against CLL patient cells (n = 9) by human NK cells of multiple genotype human donors.

Detailed description of the invention

The inventors found that anti-CD40 antibodies, such as CHIR-12.12, can mediate potential antibody-dependent cellular cytotoxicity (ADCC) of CD40-expressing target cells under conditions where other ADCC mediated antibodies are less effective or relatively incapacitated. Found. Unlike other antibodies, for example, Rituximab, an anti-CD40 antibody used in the present invention, has two FcγRIIIa amino acids 158 on natural killer (NK) cells of human patients with binding properties suitable for inducing potential ADCC. It can bind to one of the allotypes (V or F). This finding was unexpected and advances our ability to treat cancer and precancerous conditions that cross one side of the entire patient.

Thus, anti-CD40 antibodies such as CHIR-12.12 are heterozygous or homozygous for FcγRIIIa-158F (genotype V / F or F / F), further homozygous for FcγRIIIa-158V (genotype V / V) can be used for the treatment of cancer and precancerous conditions associated with CD40-expressing cells of a human patient.

The present invention thus provides a method of treating a cancer or precancerous condition associated with CD40-expressing cells of a human patient, wherein said human patient is FcγRIIIa-158F heterozygous or homozygous (genotype V / F or F / F) And administering a therapeutically or prophylactically effective amount of an anti-CD40 antibody to said human patient. The invention also relates to anti-CD40 antibodies in the manufacture of a medicament for the treatment of cancer or precancerous conditions involving CD40-expressing cells in human patients who are heterozygous or homozygous for FcγRIIIa-158F (genotype V / F or F / F). It provides the use of a therapeutically or prophylactically effective amount of.

As mentioned above, the clinical activity of Rituximab in NHL has been shown to correlate with the FcγRIIIa genotype of patients.

Patients with FcγRIIIa 158aa polymorphism of F / F are less sensitive to rituximab than patients with V / V or V / F (eg, Cartron et al. (2002) Blood 99 (3): 754-758 Or Dall'Ozzo et al. (2004) Cancer Res. 64: 4664-4669). Thus, the present invention is particularly advantageous for the treatment of cancer and precancerous conditions that are not sensitive to the treatment of anti-CD20 antibodies such as Rituximab.

Anti-CD40 antibodies such as CHIR-12.12 are heterozygous or homozygous for FcγRIIIa-158F (genotype V / F or F / F), further homozygous for FcγRIIIa-158V (genotype V / V) It can be used in a method of inhibiting antibody production by B cells in a human patient.

Thus, the present invention provides a method for preparing a heterozygous or homozygous (genotype V / F or F / F) for FcγRIIIa-158F, comprising administering to a human patient an effective amount of an anti-CD40 antibody, such as CHIR-12.12. Provided are methods for inhibiting antibody production by B cells. The invention also provides the use of an effective amount of an anti-CD40 antibody in the manufacture of a medicament for inhibiting antibody production by B cells in a human patient who is heterozygous or homozygous for FcγRIIIa-158F (V / F or F / F). do.

This was not expected by those skilled in the art to be able to inhibit antibody production by B cells in human patients who are heterozygous or homozygous for FcγRIIIa-158F (genotype V / F or F / F).

The present invention allows the treatment to be selected for individual human patients based on the FcγRIIIa-158 genotype of the patient by ADCC-mediated anti-CD40 antibody administration.

The present invention provides methods for identifying human patients with cancer or precancerous conditions treatable with anti-CD40 antibodies that are resistant to the treatment of Rituximab (Rituxan®), including:

a) identifying a human patient with a cancer or precancerous condition associated with CD40-expressing cells that is resistant to the treatment of Rituximab (Rituxan®); And

b) determining the FcγRIIIa-158 genotype (V / V, V / F or F / F) of the human patient;

Wherein when the human patient is heterozygous or homozygous for FcγRIIIa-158F (genotype V / F or F / F), the cancer or precancerous condition can be treated with an anti-CD40 antibody. The invention may further comprise administering a therapeutically or prophylactically effective amount of an anti-CD40 antibody to a human patient identified using this method.

This method of identifying human patients with cancer or precancerous conditions treatable with anti-CD40 antibodies can be readily performed by those skilled in the art using suitable diagnostic kits. The kit includes reagents suitable for determining the FcγRIIIa-158 genotype of a human patient. Accordingly, the present invention also provides kits for identifying human patients with cancer or precancerous conditions treatable with an anti-CD40 antibody, comprising reagents for determining the FcγRIIIa-158 genotype of a human patient. Suitable kits are described in more detail elsewhere herein.

The invention also provides a method of selecting antibody therapy for the treatment of human patients with cancer or precancerous conditions that are resistant to the treatment of Rituximab (Rituxan®), including:

a) identifying a human patient with a cancer or precancerous condition associated with CD40-expressing cells that is resistant to the treatment of Rituximab (Rituxan®); And

b) determining the FcγRIIIa-158 genotype of said human patient (V / V, V / F or F / F);

Wherein the human patient is heterozygous or homozygous for FcγRIIIa-158F (genotype V / F or F / F), the anti-CD40 antibody is selected for treatment of the cancer or precancerous condition. In particular, anti-CD40 antibodies may be selected prior to treatment with Rituximab (Rituxan®). The invention may further comprise administering a therapeutically or prophylactically effective amount of an anti-CD40 antibody to a human patient identified using this method.

This method of selecting antibody therapy for the treatment of human patients with cancer or precancerous conditions can be readily performed by those skilled in the art using suitable diagnostic kits. The kit should contain a reagent suitable for determining the FcγRIIIa-158 genotype of a human patient. Accordingly, the present invention also provides kits for the selection of antibody therapy for the treatment of human patients with cancer or precancerous conditions associated with CD40-expressing cells, comprising reagents for determining the FcγRIIIa-158 genotype of human patients. .

The inventors have surprisingly found that anti-CD40 antibodies, such as CHIR-12.12, are not significantly internalized by CD40-expressing cells after administration. Instead, anti-CD40 antibodies, such as CHIR-12.12, were distributed substantially uniformly on the surface of CD40-expressing cells after a significant time after administration. In contrast to other antibodies, this is in particular an anti-CD20 antibody such as Rituximab (Rituxan®).

The duration of CD40 binding on the surface of CD40-expressing cells and the uniform distribution of anti-CD40 antibodies on the surface of CD40-expressing cells suggest that CD40 through binding of the anti-CD40 antibody to FcRs, such as FcγRIIIa, on NK cells. -Mediates potential antibody-dependent cellular cytotoxicity (ADCC) of expression target cells.

Accordingly, the present invention provides a method of treating a cancer or precancerous condition associated with CD40-expressing cells of a human patient, the method comprising administering to said human patient a therapeutically or prophylactically effective amount of an anti-CD40 antibody. As such, anti-CD40 antibodies are not significantly internalized by CD40-expressing cells after administration.

The present invention provides a method of treating a cancer or precancerous condition associated with a CD40-expressing cell of a human patient, the method comprising administering to said human patient a therapeutically or prophylactically effective amount of an anti-CD40 antibody. Anti-CD40 antibodies are distributed substantially uniformly on the surface of CD40-expressing cells after administration.

The invention also provides a method of treating a cancer or precancerous condition associated with a CD40-expressing cell of a human patient, the method comprising administering an anti-CD40 antibody to the human patient, the method of treatment of an anti-CD40 antibody. Or a prophylactically effective amount is present on the surface of CD40-expressing cells in said human patient after administration.

This feature of the invention thus relates to the administration of slow-internalized antibodies to the patient. "Low-internalization antibody" means an antibody that remains on the cell surface for a significant period of time. As will be appreciated by those skilled in the art, this property contrasts with properties that are considered advantageous for many of the therapeutic applications actually required for the internalization of antibody-receptor complexes for effective therapy. In this context, the time period of substantial period is generally 3 hours, preferably 6 hours, more preferably 12 hours, more preferably 24 hours, 36 hours, 48 hours, 72 hours, 96 hours, 120 hours, 144 hours. Exceed 168 hours or more.

Preferably, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more CD40-expressing Antibodies initially placed on the surface of the cell remain on the cell surface after a significant period of time as described above.

Internalization of antibodies can be assessed by various assays. For example, cell lines such as the Daudi lymphoma cell line, or ARH77 MM cell line, can be used to assess the effect of antibody binding candidates on internalization. Cells are incubated with human IgG1 (control antibody) or candidate antibody on ice (addition of 0.1% sodium azide for blocking internalization) or at 37 ° C. (no sodium azide) for a period of time, preferably 3 hours. It became.

After washing with cold stain buffer (eg PBS + 1% BSA + 0.1% sodium azide), the cells were stained. For example, it is stained on ice for 30 minutes with goat anti-human IgG-FITC. The degree of staining can then be assessed; In this example, the geometric mean fluorescence intensity (MFI) could be recorded, for example, by a FACS Caliber. Other suitable measurements are known to those skilled in the art (eg, http://www.abgenix.com/documents/SBS2003%20poster.pdf).

In the experiments described in Examples 4 and 5 herein, no differences in MFI were observed between cells cultured with CH12.12 in the presence of sodium azide or on sodium azide deficient on ice ( 7-10). These data indicate that CH12.12, when bound to CD40, is not internalized and continues to be expressed on the cell surface for longer than rituximab.

An overview of the classification methods and procedures that can be used to utilize the present invention is given below. It is to be understood that the invention is not limited to the particular methodology, protocols, cell lines, vectors and reagents presented. It is also to be understood that the terminology herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention. It is intended that the scope of the invention only be limited by the appended claims.

Standard abbreviations for nucleotides and amino acids are used herein.

The practice of the present invention will, unless otherwise indicated, employ conventional techniques of molecular biology, microbiology, recombinant DNA technology and immunology, which are within the scope of those skilled in the art.

Such techniques are explained fully in the literature. Examples of textbooks that are particularly suitable for consultation include: Sambrook et al. (1989) Molecular Cloning; A Laboratory Manual (2d ed.); D.N G1over, ed. (1985) DNA Cloning, Volumes I and II; M.J. Gait, ed. (1984) Oligonucleotide Synthesis; B.D. Hames & SJ. Higgins, eds. (1984) Nucleic Acids Hybridization; B.D. Hames & S.J. Higgins, eds. (1984) Transcription and Translation; R.I. Freshney, ed. (1986) Animal Cell Culture; Immobilized Cells and Enzymes (IRL Press, 1986); B. Perbal (1984) A Practical Guide to Molecular Cloning; the Methods in Enzymology series (Academic Press, Inc.), especially volumes 154 &155; J.H. Miller and M.P. Calos, eds. (1987) Gene Transfer Vectors for Mamalian Cells (Cold Spring Harbor Laboratory); Mayer and Walker, eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); Scopes (1987) Protein Purification: Principles and Practice (2d ed .; Springer Verlag, N.Y.); And D.M. Weir and C. C. Blackwell, eds. (1986) Handbook of Experimental Immunology, Volumes I-IV.

The methods of the invention relate to the use of anti-CD40 antibodies in the treatment of cancer and precancerous conditions associated with CD40-expressing cells.

"CD40", "CD40 antigen", or "CD40 receptor" refers to the transmembrane glycoproteins of the tumor necrosis factor (TNF) receptor family of 50-55 kDa (eg, US Pat. Nos. 5,674,492 and 4,708,871) Stamenkovic et al. (1989) EMBO 8: 1403; Clark (1990) Tissue Antigens 36:33; Barclay et al (1997) The Leucocyte Antigen Facts Book (2d ed .; Academic Press, San Diego). Two species of isoforms of human CD40 encoded by alternatively spliced transcriptional variants of this gene were identified. The first isoform (also known as "long isoform" or "isoform 1") is expressed as a 277-amino acid precursor polypeptide (SEQ ID NO: 9; originally reported as GenBank Accession Number CAA43045, GenBank Accession Number NP_001241 Identified as isotype 1, which is encoded by SEQ ID NO: 8 (GenBank Accession Nos. X60592 and NM_001250) and bears the signal sequence set forth by the first 19 residues. The second isoform (also known as "short isoform" or "isotype 2") is expressed as a 203-amino acid precursor polypeptide (SEQ ID NO: 7; GenBank Accession No. NP_690593), which is SEQ ID NO: 6 (GenBank Accession No. NM_152854) and also carries the signal sequence set forth by the first 19 residues. The two isoform precursor polypeptides of this human CD40 share the first 165 residues (ie, 1-165 residues of SEQ ID NO: 7 and SEQ ID NO: 9). Short isoform precursor polypeptides (denoted by SEQ ID NO: 7) are encoded by transcriptional variants lacking the coding moiety (SEQ ID NO: 6) and induce translation frame shifts; The resulting CD40 isoform contains a shorter C-terminus (166-203 residues of SEQ ID NO: 7) that is distinct from that contained in the long isoform of CD40 (shown at 166-277 residues of SEQ ID NO: 9). C-terminal). For the purposes of the present invention, the terms "CD40," or "CD40 antigen," "CD40 cell surface antigen" or "CD40 receptor" encompass both short and long isoforms of CD40. CD40 antigen was glycosylated in whole or in part.

As noted elsewhere herein, CD40 is a combination of both normal and neoplastic human B cells, dendritic cells, monocytes, macrophages, CD8 + T cells, endothelial cells, mononuclear and epithelial cells, and lung, breast, ovary, bladder And on the surface of many solid tumors, including colon cancer. Malignant B cells of the B-cell family of tumor types express CD40, which seems to depend on CD40 signals for survival and proliferation. Transgenic cells of patients with low and high B-cell lymphoma, B-cell acute lymphoblastic leukemia, multiple myeloma, chronic lymphocytic leukemia, Waldenstrom's macroglobulinemia, and Hodgkin's disease express CD40. CD40 expression is also detected in acute myeloid leukemia and 50% of AIDS-associated lymphomas. Although many carcinomas and sarcomas show high levels of CD40 expression, the role of CD40 signals on CD40 expression on these cancer cells is not well known. CD40-expressing carcinomas include bladder carcinoma, breast carcinoma, prostate cancer, renal cell carcinoma, undifferentiated nasopharyngeal carcinoma (UNPC), squamous cell carcinoma (SCC), thyroid papillary carcinoma, cutaneous malignant melanoma, gastric carcinoma, and liver carcinoma do.

By "CD40-expressing cell" is meant herein all normal or malignant cells that express a detectable level of CD40 antigen. Preferably, the CD40-expressing cell is a cell expressing a detectable level of cell-surface CD40 antigen. Methods for detecting CD40 expression in cells are well known in the art and are not limited to PCR techniques, immunohistochemistry, flow cytometry, western blots, ELISAs, and the like. This method enables the detection of CD40 mRNA, CD40 antigen and cell-surface CD40 antigen. Detection of cell-surface CD40 expression can be performed as described in Example 3 herein or by other suitable methods.

The malignant cell may be a malignant B cell. “Malignant B cells” are lymphomas including low, moderate, and high B cell lymphomas, immune blast lymphomas, non-Hodgkin's lymphomas, Hodgkin's disease, Epstein-Barr virus (EBV) -induced lymphomas, and AIDS-related lymphomas. B cell and B cell derived from means all tumorous B cells, including but not limited to acute lymphoblastic leukemia, myeloma, chronic lymphocytic leukemia, and the like.

"CD40 ligand" or "CD40L" means 32-33 kDa transmembrane proteins that exist primarily in two small biologically active soluble forms, 18 kDa and 31 kDa, respectively (Graf et al (1995) Eur. J. Immunol. 25: 1749-1754; Mazzei et al. (1995) J Biol. Chem. 270: 7025-7028; Pietravalle et al. (1996) J Biol Chem. 271: 5965-5967). Human CD40L is known as CD154 or gp39. "CD40 ligand" or "CD40L" refers to any other peptide, polypeptide, or protein capable of binding to and activating one or more CD40 signaling pathways. Thus, "CD40 ligands" include, but are not limited to, full length CD40 ligand proteins and variants, and fragments thereof that possess sufficient activity to perform CD40 signal stimulation on CD40-expressing cells. Modifications of natural CD40 ligands, such as human CD40L, include, but are not limited to, substitutions, deletions, cleavage, extension, fusion proteins, fragments, peptidomimetics, and the like.

"CD40 signal" means all biological activity exhibited by the interaction of cell-surface CD40 with a CD40 ligand or other agent, such as an agent antibody. Examples of CD40 signals are signals that induce proliferation and survival of CD40-expressing cells and stimulation of one or more CD40-signal pathways in CD40-expressing cells. CD40 "signal pathways" or "signal pathways" are reactions of CD40 receptors and CD40 ligands, eg, CD40L, which, when transmitted through the signal pathway, are one or more in the molecular signal cascade. At least one biochemical reaction, or group of biochemical reactions, that produces a signal that induces activation of a downstream molecule. Signal transduction pathways involve a plurality of signal transduction molecules that induce signal transmission from cell-surface CD40 receptors to the cell nucleus through one or more series of signal transduction molecules, for example, through the cytoplasm of a cell, through the cell's plasma membrane. It is associated with signal transduction molecules. Subjects of the invention are in particular the CD40 signaling pathway, which induces activation of AKT and ultimately activates NF-κB via the NF-κB signal pathway; And a mitogen-activated protein kinase (MAPK) signal pathway that includes the MEK / ERK signal pathway and the MEK / p38 signal pathway, and induces activation of ERK and p38, respectively.

As described above, the present invention provides a method for treating a cancer or precancerous condition associated with CD40-expressing cells of a human patient, wherein the human patient is heterozygous or homozygous for FcγRIIIa-158F (genotype V / F or F / F), and the method comprises administering to said human patient a therapeutically or prophylactically effective amount of an anti-CD40 antibody.

"Human patient" means a human patient at risk of developing or recurring, who is suffering from a cancer or precancerous condition associated with all CD40-expressing cells.

"Cancer or precancerous condition associated with CD40-expressing cells" means all B-cell lineage cancers, non-B cell hematologic malignancies, and solid tumors known to be associated with CD40-expressing cells.

The methods of the invention are useful for the therapeutic treatment of cancer of the B-cell family. B-cell lineages associated with CD40-expressing cells include, but are not limited to, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), prelymphocytic leukemia (PLL), bovine lymphocytic leukemia ( SLL), hairy cell leukemia, Hodgkin's disease, multiple myeloma, Valdanstrom's macroglobulinemia, heavy chain disease, and extensive bovine lymphoid lymphoma, vesicles, DLBCL, mucosal related lymphoid tissue lymphoma, monocyte B cell lymphoma, spleen Lymphomas, including but not limited to lymphomas, lymphoma granulomatosis, endovascular lymphoma, immune blast lymphoma, AIDS-associated lymphoma, and the like.

Thus, the methods of the present invention have found use for the treatment of subjects with non-Hodgkin's lymphoma associated with abnormal, non-inhibitable B cell proliferation or accumulation. For the purposes of the present invention, such lymphomas will be queried according to the Working Formulation Classification Scheme and are low, moderate, and highly classified B cell lymphomas ("The Non-Hodgkin's Lymphoma Pathologic Classification Project," Cancer 49 (1982): 2112-2135). Thus, low B cell lymphomas include bovine lymphoid, vesicular bovine-dividing cells, and vesicle mixed bovine-dividing and giant cell lymphomas; Moderate lymphomas include vesicle giant cells, broad bovine divide cells, broad mixed bovine cells and giant cells, and diffuse giant cell lymphoma; High lymphomas include giant cell immune blasts, lymphoblastic, and bovine non-dividing cell lymphomas of the bucket and non-bucket types.

The methods of the invention are useful for the therapeutic treatment of B cell lymphomas classified by the Revised European and US Lymphoma Classification (REAL) system. Such B cell lymphomas include, but are not limited to, precursor B cell neoplasias such as B lymphoblastic leukemia / lymphoma; B-cell chronic lymphocytic leukemia / small lymphocytic lymphoma, lymphoid cytoplasmic lymphoma / immunoma, mantle cell lymphoma (MCL), vesicle central lymphoma (vesicle) (broad small cells, broad mixed small cells and giant cells, and diffuse large cells Cell lymphoma), marginal B cell lymphoma (including extranodular, nodule, and spleen types), plasmacytoma / myeloma, diffuse giant cell B cell lymphoma of subtype primary mediastinum (thymus), Burkitt lymphoma, and Burkitt-like elevation Peripheral B cell neoplasia including B cell lymphoma; And unclassified low or high B cell lymphoma.

The method of the present invention is useful for the therapeutic treatment of a precancerous condition known as MGUS (a monoclonal gamma globulin with no significance). About 25% of patients with MGUS eventually develop multiple myeloma (MM) or related plasma cell disease (Kyle (1993) Mayo Clinic. Proc. 68: 26-36). Proliferation of malignant plasma cells in the bone marrow, detection of serum or urine monoclonal protein (M protein), anemia, hypercalcemia, renal failure, and osteolysis are clinical findings of MM, whereas MGUS is accompanied by other clinical characteristics of MM. And clinically recognized as the presence of M protein in serum or urine (eg, Kyle and Lust (1989) Semin. Hematol 26: 176-200; Greipp and Lust Stem Cells (1995) 13: 10-21 ). MGUS patients are asymptomatic and have a stable measurement of M protein (Kyle (1993) Mayo Clinic. Proc. 68: 26-36). When MGUS is identified in a subject, maintenance therapy with a suitable anti-CD40 antibody, eg, antagonist anti-CD40 antibody, blocks the development of multiple myeloma in such patients.

The methods of the invention are also useful for therapeutic treatment of non-B cell-associated hematologic malignancies associated with CD40-expressing cells, such as acute myeloid leukemia.

The methods of the invention are also useful for the therapeutic treatment of solid tumors. Solid tumors associated with CD40-expressing cells include ovarian cancer, lung cancer (eg, non-small cell lung cancer of squamous cell carcinoma, adenocarcinoma, and giant cell carcinoma types, and small cell lung cancer), breast cancer, colon cancer, kidney cancer (eg , Renal cell carcinoma), bladder cancer, liver cancer (including hepatocellular carcinoma), gastric cancer, cervical cancer, prostate cancer, nasal pharyngeal cancer, thyroid cancer (eg thyroid papillary carcinoma), skin cancer such as melanoma And sarcomas, including, for example, osteosarcomas and Ewing's sarcomas.

The cancer or precancerous condition associated with the CD40-expressing cell may be a cancer or precancerous condition in which CD40 signaling on the CD40-expressing cell is an undesirable level or the cancer or precancerous condition may be indirectly related to the CD40-expressing cell. "A cancer or precancerous condition at which CD40 signal is undesirable" means a cancer or precancerous condition associated with a CD40 signal at which onset or progression is undesirable.

"Undesirable level of CD40 signal" means any physiologically undesirable level of CD40 signal that occurs in CD40-expressing cells of human patients with cancer or precancerous conditions.

The cancer or precancerous condition may be a cancer or precancerous condition associated with CD20-expressing cells. Such cancer or precancerous conditions include, but are not limited to, the B cell malignancies mentioned elsewhere herein.

The present invention is particularly advantageous for cancer and precancerous conditions associated with cells expressing both CD40 and CD20. This is because the novel uses of anti-CD40 antibodies, such as CHIR-12.12, disclosed herein address the problems associated with the use of anti-CD20 antibodies, such as Rituxan®. In particular, as described in detail elsewhere herein, the present invention relates to anti-CD20 antibodies, such as Rituxan®, for patients who are homozygous or heterozygous (genotype V / F or F / F) to FcγRIIIa-158F. Resistant to therapy with other tumor therapies, including, treatment of patients with cancer or precancerous conditions is possible.

In the therapeutic methods of the present invention, at least one anti-CD40 antibody as defined herein is used for enhancing a positive therapeutic response to cancer or precancerous conditions.

"Positive therapeutic response" to a cancer or precancerous condition means an improvement in a cancer or precancerous condition associated with the therapeutic activity of an anti-CD40 antibody, and / or an improvement in symptoms associated with a cancer or precancerous condition. That is, antiproliferative effects, prevention of further tumor proliferation, reduction in tumor size, reduction in cancer cell numbers, and / or reduction in one or more symptoms associated with CD40-expressing cells. Thus, for example, a positive therapeutic response means one or more of the following improvements in disease: (1) reduction in tumor size; (2) reduction in the number of cancerous (ie, neoplastic) cells; (3) increased tumor cell death; (4) inhibition of neoplastic cell survival; (4) inhibition of tumor growth (ie slowing to a certain extent, preferably stopping); (5) inhibition of infiltration of cancer cells into peripheral organs (ie, slowing to a certain extent, preferably stopping); (6) inhibition (ie, slowing to a certain extent, preferably stopping) tumor metastasis; (7) prevention of further tumor proliferation; (8) increased patient survival; And (9) to some extent alleviate one or more cancer-related symptoms.

Positive therapeutic response within a given malignancy can be measured by standardized response criteria specific for the malignancy. Tumor responses include screening techniques such as magnetic resonance imaging (MRI) scans, x-ray imaging, computed tomography (CT) scans, bone scan imaging, endoscopic examination, and bone marrow aspiration (BMA) and counting tumor cells in circulation Tumor biopsy sampling can be used to assess tumor morphological changes (ie, total tumor load, tumor size, etc.). In addition to this positive therapeutic response, subjects who have undergone treatment with anti-CD40 therapeutics may experience beneficial effects in improving disease related symptoms. Thus, in B cell tumors, a subject may experience so-called B symptoms, namely cold, fever, weight loss, and / or a decrease in urticaria. In precancerous conditions, therapy with anti-CD40 therapeutics can block and / or prolong the pre-onset time of multiple myeloma in a subject suffering from a related malignant condition, eg, MGUS.

Improvement of the disease is characterized by a complete response. "Complete response" means the absence of clinically detectable disease with normalization of cerebrospinal fluid (CSF) or abnormal monoclonal protein for all previous abnormal radiological studies, bone marrow, and myeloma. Such a response should last at least 4-8 weeks after treatment with the method of the present invention, or in certain diseases, sometimes 6-8 weeks. In other embodiments, amelioration of the disease can be classified as a partial response. A “partial response” is at least about 50% reduction in all measurable tumor loads (ie, the amount of malignant cells present in a subject, or the measured volume or abnormal monoclonal protein in a tumor volume) and as needed According to 4 to 8 weeks of duration. For myeloma, a normal response (25-50% reduction of myeloma protein in urine) is also considered a response. This response applies only to measurable tumors.

A "therapeutically or prophylactically effective dose" or "therapeutically or prophylactically effective amount" means an anti-positive response to treatment of patients with cancer or precancerous conditions associated with CD40-expressing cells when administered. Means the amount of CD40 antibody. Suitable dosages are described in more detail herein. The method of treatment may comprise a single dose or multiple doses of a therapeutically effective dose of an anti-CD40 antibody, as described in detail herein.

As used herein, "tumor" refers to all tumorous cell growth and proliferation, whether malignant or benign and all precancerous and cancerous cells and tissues. As used herein, “tumorality” refers to any form of unregulated or unregulated cell growth that indicates abnormal tissue growth, whether malignant or benign. Thus, "tumor cells" include malignant and benign cells with unregulated or unregulated cell growth.

The terms "cancer" and "cancerous" are or describe the physiological state of a mammal generally characterized by unregulated cell growth. Examples of cancers include, but are not limited to, lymphomas and leukemias, and solid tumors. "B cell-associated cancer" or "B-cell family cancer" means any type of cancer in which unregulated or unregulated cell growth is associated with B cells.

"Treatment" is used herein to treat, cure, alleviate, alleviate, alter, treat, ameliorate a disease, symptom of disease, or disposition for a disease, a symptom of disease, a symptom, or disposition for a disease, For the purpose of improving or affecting, it is defined as applying or administering an anti-CD40 antibody to a patient, or applying or administering an anti-CD40 antibody to a tissue or cell line isolated from a patient. A "treatment" also includes a pharmaceutical composition comprising an anti-CD40 antibody for the purpose of treating, healing, alleviating, alleviating, altering, treating, improving, improving, or affecting a disease, symptom of a disease, or propensity for a disease. Means to apply or administer to a patient having a disease, symptom of disease, or disease propensity, or to apply or administer a pharmaceutical composition comprising an anti-CD40 antibody to a tissue or cell line isolated from the patient.

"Anti-tumor activity" refers to a decrease in the rate of malignant CD40-expressing cell proliferation or accumulation, and thus a reduction in growth rate of existing tumors or tumors growing during therapy, and / or existing tumorous (tumor) cells or newly formed species. Disruption of benign cells, and consequently a reduction in tumor overall size during therapy. Therapy with at least one anti-CD40 antibody elicits a beneficial physiological response to the treatment of disease states related to CD40 signal stimulation on human CD40-expressing cells.

The methods of the invention are particularly useful for the treatment of cancer and precancerous conditions, including those listed above, which are resistant to foremost oncology treatment. "Tumor therapy" means all cancer treatments such as chemotherapy, surgery, radiation therapy, anti-cancer antibody therapy, and combinations thereof. Examples of oncology treatments are described in detail elsewhere herein. "Resistant" means that a particular cancer is resistant or non-responsive to therapy with a particular tumor therapeutic agent. The cancer may be specific at the start of treatment with a particular therapeutic agent (ie non-responsive to initial exposure of the therapeutic agent) or as a result of development of resistance to the therapeutic agent during the first treatment period with the therapeutic agent or during subsequent treatment with the therapeutic agent. May be resistant to therapy with the therapeutic agent. Accordingly, the present invention is useful for the treatment of human patients who are resistant to therapy with anticancer agents, if the human patient is resistant to therapy with anticancer agents or is non-responsive.

The method of the invention relates to the use of anti-CD40 antibodies. An "antibody" is generally a heterotetra glycoprotein of about 150,000 Daltons and consists of two identical light chains (L) and two identical heavy chains (H). Each light chain is bound to the heavy chain by one covalent disulfide bond, and the number of disulfide bonds is different for the heavy chains of different immunoglobulin isotypes. Each heavy and light chain is regularly filled with interchain disulfide bridges. Each heavy chain has at one end a variable domain (VH) linked to a number of constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at the other end; The constant domain of the light chain is aligned with the first constant domain of the heavy chain and the light chain variable domain is aligned with the variable domain of the heavy chain. Certain amino acid residues are known to form an interface between the light and heavy chain variable domains. The term "variable" refers to the fact that certain parts of the variable domains differ widely in sequence between antibodies. The variable region confers antigen-binding specificity. The constant domains are not directly involved in the binding of the antibody to antigens, but include, for example, Fc receptor (FcR) binding, antibody involvement in antibody-dependent cellular cytotoxicity, initiation of complement dependent cytotoxicity, and mast cell degranulation. It works as an effector.

The “light chains” of antibodies of the vertebrate species (immunoglobulins) are assigned to two distinctly distinct types called kappa (K) and lambda (λ) based on the amino acid sequences of their constant domains.

Depending on the amino acid sequences of the constant domains of their "heavy chains", immunoglobulins can be assigned to different classes. There are five main classes of human immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which may be further divided into subclasses (isotypes), eg, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. Subunit structures and three-dimensional conformation of other classes of immunoglobulins are well known. Different isotypes have different effector functions. For example, human IgG1 and IgG3 isotypes possess ADCC (antibody dependent cell-mediated cytotoxicity) activity. IgG1 antibodies, in particular human IgG1 antibodies, are particularly useful in the methods of the invention.

A "human working cell" is a white blood cell that expresses one or more FcRs and performs effector functions. Preferably, the cells express at least FcγRIII and perform an antigen-dependent cell-mediated cytotoxicity (ADCC) effector function. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, macrophages, eosinophils, and neutrophils, with PBMCs and NK cells being preferred. Antibodies with ADCC activity are generally of the IgG1 or IgG3 isotype. It is noted that with the isolation of IgG1 and IgG3 antibodies, such ADCC-mediated antibodies can be prepared by genetic engineering of the variable regions of non-ADCC antibodies or variable region fragments into IgG1 or IgG3 isotype constant regions.

The term "Fc receptor" or "FcR" is used to describe a receptor that binds to the Fc region of an antibody. Preferred FcRs are naturally-sequence human FcRs. Moreover, preferred FcRs bind to IgG antibodies (gamma receptors) and include allelic variants, receptors of the FcγRI, FcγRII, and FcγRIII subclasses which include spliced forms of these receptors. FcγRII receptors include FcγRIIA (“activating receptor”) and FcγRIIB (“inhibiting receptor”), whose cytoplasmic domains primarily carry other similar amino acid sequences. Activating receptor FcγRIIA contains immunoreceptor tyrosine-based activation motifs (ITAMs) in their cytoplasmic domains. Inhibitory receptors FcγRIIB contain an immunoreceptor tyrosine-based inhibitory motif (ITIM) in their cytoplasmic domain (Daeron (1997) Annu. Rev. Immunol. 15: 203-234). FcRs are described in Ravetch and Kinet (1991) Annu. Rev. Immunol. 9: 457-492 (1991); Capel et al. (1994) Immunomethods 4: 25-34; And de Haas et al. (1995) J Lab. Clin. Med. 126: 330-341. Other FcRs, including those to be identified in the future, are encompassed by the term "FcR" herein. The term includes the neonatal receptor, FcRn, responsible for delivering maternal IgG to the fetus (Guyer et al. (1976) J. Immunol. 117: 587 and Kim et al. (1994) J Immunol. 24: 249 (1994)).

The term “antibody” is used herein in its broad sense and refers to antibody fragments (eg, Fab, F (ab ') ₂ , Fv, and other fragments that possess the ability to specifically bind to all bound antibodies, CD40 antigens. ), Single chain antibodies, dibodies, antibody chimeras, hybrid antibodies, bispecific antibodies, humanized antibodies, and the like), and recombinant peptides including those described above. The term "antibody" includes both polyclonal and monoclonal antibodies.

As used herein, "anti-CD40 antibody" encompasses all antibodies that specifically recognize the CD40 antigen. In one embodiment of the invention, the anti-CD40 antibodies, in particular monoclonal anti-CD40 antibodies, used in the methods of the invention exhibit a strong single-site binding affinity for the CD40 antigen. Such monoclonal antibodies have at least 10 ⁻⁵ M, at least 3 × 10 ⁻⁵ M, preferably at least 10 ⁻⁶ M, or at least 10 ⁻⁷ M, more preferably as measured using standard assays such as Biacore ™. Affinity (K _D ) for at least 10 ⁻⁸ M, or at least 10 ⁻¹² M, to CD40. Biacore analysis is well known in the art and details are presented in the "BIAapplications handbook". The method described in WO 01/27160 can be used to adjust binding affinity.

"Specifically recognize" or "specifically bind" means that the anti-CD40 antibody does not bind to an irrelevant antigen, such as a CD20 antigen.

In one embodiment of the invention, the anti-CD40 antibodies, in particular monoclonal antibodies, used in the methods of the invention exhibit strong binding affinity for human FcγRIIIa-158V. Preferably, the anti-CD40 antibody used in the methods of the invention binds to human FcγRIIIa-158V with an affinity (K _D ) of at least about 0.5 μM when measured by standard assays such as Biacore ™. As described in Example 6 herein, the CHIR-12.12 antibody binds to human FcγRIIIa-158V with an affinity (K _D ) of 492 nM.

In one embodiment of the invention, the anti-CD40 antibody, in particular monoclonal antibody, used in the methods of the invention exhibits a strong binding affinity for human FcγRIIIa-158F. Preferably, the anti-CD40 antibody used in the methods of the invention binds to human FcγRIIIa-158F with an affinity (K _D ) of at least about 12 μM when measured by standard assays such as Biacore ™. Preferably, the anti-CD40 antibody used in the methods of the invention has an affinity of at least about 10 μM, at least about 8 μM, at least about 6 μM, at least about 5 μM, at least about 4 μM, or at least about 3 μM ( K _D ) binds to human FcγRIIIa-158F. As described in Example 6 herein, the CHIR-12.12 antibody binds to human FcγRIIIa-158F with an affinity (KD) of 2.8 μM.

In one embodiment of the invention, the anti-CD40 antibodies, in particular monoclonal antibodies, used in the methods of the invention exhibit strong binding affinity for both human FcγRIIIa-158V and FcγRIIIa-158F. Preferably, the anti-CD40 antibody used in the methods of the invention binds to human FcγRIIIa-158V with an affinity (KD) of at least about 0.5 μM and at least about 12 μM when measured by standard assays such as Biacore ™ Binds to human FcγRIIIa-158F with an affinity of (KD).

Antibodies used in the methods of the invention can be produced using any suitable antibody production method known to those skilled in the art.

The anti-CD40 antibody used in the methods of the invention may be a polyclonal antibody. Thus, polyclonal sera can be prepared using conventional methods. In general, a solution containing the antigen of interest (in this case the CD40 antigen) is first used to immunize a suitable animal, preferably a mouse, rat, rabbit, or goat. Rabbits or goats are preferred for the preparation of polyclonal sera, due to the volume of serum available and the availability of labeled anti-rabbit and anti-chlorine antibodies.

Sera of immunized animals can be selected for antibody reactivity to the initial antigen. Lymphocytes can be isolated from lymph nodes or pancreatic cells and can be further selected for B cells by selecting CD138-negative and CD 19-positive cells. In one aspect of the invention, such B cell culture (BCC) can be fused with myeloma cells to produce the hybridomas described in detail herein.

Polyclonal sera can also be prepared in transgenic animals, preferably in mice comprising human immunoglobulin loci. In a preferred embodiment of the invention, Sf9 cells expressing the protein of interest (in this case the CD40 antigen) can be used as an immunogen. Vaccination involves mixing or emulsifying the antigen-containing solution in saline, preferably in an adjuvant such as Freund's complete adjuvant and injecting the mixture or emulsion parenterally (generally in the subcutaneous tissue or muscle). Is performed. Doses of 50-200 μg / injection are generally sufficient. Vaccination is usually elevated 2-6 weeks after one or more infusions using protein saline solution, preferably Freund's incomplete adjuvant. In another embodiment, antibodies are generated by in vitro vaccination using what is known in the art, which is considered equivalent to in vivo vaccination for the purposes of the present invention. Polyclonal antisera can be obtained by bleeding immunized animals into glass or plastic containers, incubating blood at 25 ° C. for 1 hour and then incubating at 4 ° C. for 2-18 hours. . Serum is recovered by centrifugation (eg 1,000 × g, 10 minutes). About 20-50 ml / bleed can be obtained from rabbits.

Sf9 ( Spodoptera production of frugiperda ) cells is disclosed in US Pat. No. 6,004,552, which is incorporated herein by reference. In the case of CD40, briefly, the sequence encoding human CD40 is recombined into baculovirus using a transfer vector. Plasmids are transfected into Sf9 cells with wild type baculovirus DNA. Recombinant baculovirus-infected Sf9 cells were identified and isolated by cloning.

Anti-CD40 antibodies used in the methods of the invention may be monoclonal antibodies. The term "monoclonal antibody" (and "mAb") as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, ie, including individual populations except naturally occurring possible mutations present in small amounts. The antibodies of are identical. This term places no limitations on the species of the antibody and is not necessary for the production of the antibody by any particular method.

In contrast to polyclonal antibody preparation, this generally includes other antibodies that are regulated against other antigenic determinants (epitopes), and each monoclonal antibody is regulated against a single determinant (epitopes) on the antigen.

"Epitope" as part of an antigen molecule, means that an antibody is produced and bound. An epitope can be a linear amino acid residue (ie, a sequence of residues within the epitope that is sequentially arranged in linear order), a nonlinear amino acid residue ("nonlinear epitope"; such epitopes are not arranged sequentially), or both linear and nonlinear amino acid residues. It may include. Anti-CD40 monoclonal antibodies suitable for use in the methods of the invention may specifically bind to epitopes of human CD40 antigen expressed on the surface of human cells.

Monoclonal antibodies used according to the invention are described in Kohler et al. (1975) hybridoma methods first disclosed in Nature 256: 495, or by recombinant DNA methods (eg, US Pat. No. 4,816,567). Monoclonal antibodies are described, for example, in McCafferty et al. (1990) Nature 348: 552-554 (1990) and the antibody phage libraries generated using the techniques described in US Pat. No. 5,514,548. Clackson et al. (1991) Nature 352: 624-628 and Marks et al. (1991) J MoI. Biol. Phage libraries at 222: 581-597 are used to describe the isolation of murine and human antibodies, respectively. Subsequent literature describes the preparation of high affinity (nM range) human antibodies by chain shuffling (Marks et al. (1992) Bio / Technology 10: 779-783), and combination infections as a strategy for construction of very large phage libraries and In vivo recombination (Waterhouse et al. (1993) Nucleic. Acids Res. 21: 2265-2266) is described. Thus, this technique is a viable alternative to traditional monoclonal antibody hybridoma techniques for isolation of monoclonal antibodies.

In Kohler et al. In the traditional method of (1975 Nature 256: 495-496), mice are generally immunized with a solution containing the antigen. Vaccination can be carried out by mixing or emulsifying the antigen-containing solution in saline, preferably in an adjuvant such as Freund's complete adjuvant and parenterally injecting this mixture or emulsion. All vaccination methods known in the art can be used to obtain the monoclonal antibodies of the invention. After vaccination of the animal, the interest (and optionally several large lymph nodes) are removed and dissociated into single cells. Interest cells can be selected by applying the cell suspension to a plate or well coated with the antigen of interest. Antigen-specific B cell expression membrane boundary immunoglobulins bind to the plate and are not rinsed. The resulting B cells, or all dissociated interest cells, are then induced to fuse with myeloma cells to form hybridomas and cultured in selection medium. The resulting cells are plated by serial dilution and the production of antibodies that specifically bind to the antigen of interest (but not to an unrelated antigen) is measured. Selected monoclonal antibody (mAb) -secreting hybridomas may be cultured in vitro (eg, in tissue culture vessels or hollow reactors), or even in vivo (plural of mice).

As another feature of the invention, the B cell culture may be further selected by responsiveness to the initial antigen, preferably. Such screening includes enzyme-link immunoassay (ELISA) using target / antigen proteins, competitive assays using known antibodies that bind to the antigen of interest, and in vitro binding with transiently transfected CHO or other cells expressing the target antigen. do.

When anti-CD40 antibodies used in the methods of the invention are prepared using recombinant DNA methods, DNA encoding monoclonal antibodies specifically binds to conventional procedures (e.g., to genes encoding heavy and light chains of murine antibodies). By using oligonucleotide probes). The hybridoma cells described herein serve as the preferred source of such DNA. When isolated, the DNA is placed into an expression vector, which means that host cells, such as E. coli cells, Simian, that are unable to produce immunoglobulin proteins to obtain the synthesis of monoclonal antibodies in recombinant host cells. simian) transfected into COS cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells. Research literature on DNA recombinant expression encoding antibodies in bacteria is described in Skerra et al. (1993) Curr. Opinion in Immunol. 5: 256 and Phickthun (1992) Immunol. Revs. 130: 151. In other embodiments, antibodies are produced in cell lines such as CHO cell lines, which are described in US Pat. No. 5,545,403; 5,545,405; And 5,998,144; It is incorporated herein by reference. Briefly, cell lines are transfected with vectors capable of expressing light and heavy chains, respectively. By transfecting two proteins on the isolated vector, chimeric antibodies can be generated. Another advantage is the precise glycosylation of the antibody.

As used herein, “host cell” refers to a microorganism or eukaryotic cell or cell line which can be, and has been, a recipient of a recombinant vector or other delivery polynucleotide, and has been cultured as a single cell entity used therein. Progeny of infected original cells. It is to be understood that the progeny of a single cell does not need to match the morphological or genomic or total DNA complement completely with the parental cells, which is due to natural, accidental or deliberate mutation.

In one embodiment of the invention, an anti-CD40 antibody, such as CHIR-12.12, can be produced in CHO cells using the GS gene expression system (Lonza Biologies, Portsmouth, New Hampshire), which uses glutamine synthetase as a marker use. US Patent No. 5,122,464; 5,591,639; 5,658,759; 5,770,359; 5,827,739; 5,879,936; 5,891,693; And 5,981,216; Their contents are incorporated herein by reference.

Monoclonal antibodies of CD40 are known in the art. For example, McMichael, ed. (1987; 1989) section on B-cell antigens of Leukocyte Typing III and IV (Oxford University Press, New York); US Patent No. 5,674,492; 5,874,082; 5,677,165; 6,056,959; WO 00/63395; International Publication Nos. WO 02/28905 and WO 02/28904; Gordon et al. (1988) J. Immunol. 140: 1425; Valle et al. (1989) Eur. J. Immunol. 19: 1463; Clark et al. (1986) PNAS 83: 4494; Paulie et al. (1989) J Immunol. 142: 590; Gordon et al. (1987) Eur. J. Immunol. 17: 1535; Jabara et al. (199O) J Exp. Med. 172: 1861; Zhang et al (199I) J Immunol. 146: 1836; Gascan et al. (199I) J Immunol. 147: 8; Banchereau et al. (1991) Clin. Immunol. Spectrum 3: 8; And Banchereau et al. (1991) Science 251: 70; All of which are incorporated herein by reference.

As mentioned above, the term "antibody" as used herein encompasses chimeric antibodies. A “chimeric” antibody refers to an antibody most preferably derived using recombinant deoxyribonucleic acid techniques and includes both human (immunologically “related” species such as chimpanzees) and non-human components. Thus, the constant region of the chimeric antibody most preferably substantially coincides with the constant region of the natural human antibody; The variable region of the chimeric antibody is most preferably derived from a non-human source and retains the desired antigen specificity for the antigen of interest (CD40). Non-human sources can be any vertebrate source that can be used to generate antibodies against the CD40 antigen. Such non-human sources include rodents (eg, rabbits, rats, mice, etc .; eg, US Pat. No. 4,816,567, incorporated herein by reference) and non-human primates (eg, Old World monkeys, apes, etc.); For example, US Pat. Nos. 5,750,105 and 5,756,096 (incorporated herein by reference), including but not limited to.

As mentioned above, the term "antibody" as used herein encompasses a humanized antibody. “Human adaptation” is the form of an antibody that contains a minimal sequence derived from a non-human immunoglobulin sequence. In most cases, the humanized antibody is a human immunoglobulin (transmitter antibody), and the residues of the hypervariable region (complementarity determining region or CDR) of the recipient are non-human species with the desired specificity, affinity, and dose. (Donor antibody) eg replaced by residues in hypervariable regions of mice, rats, rabbits, or non-human primates. The term "complementarity determining region" refers to an amino acid sequence that together defines the binding affinity and specificity of the native Fv region of a native immunoglobulin binding site. For example, Chothia et al. (1987) J MoI. Biol. 196: 901-917; Kabat et al. (1991) U.S. Dept. of Health and Human Services, NIH Publication No. See 91-3242. The term "constant region" refers to the portion of an antibody molecule that confers effector function. In previous studies on the production of non-immunogen antibodies for use in the treatment of human disease, the mouse constant regions were replaced by human constant regions. The constant region of the subject humanized antibody is derived from human immunoglobulins. However, these humanized antibodies can induce unwanted, potentially dangerous human immune responses, and there is a loss of affinity.

Human adaptation can be performed according to the method of Winter and co-investigators by replacing rodent or mutant rodent CDRs or CDR sequences with the corresponding sequences of human antibodies (Jones et al. (1986) Nature 321: 522-525; Riechmann et al. (1988) Nature 332: 323-327; Verhoeyen et al. (1988) Science 239: 1534-1536). See also US Patent Nos. 5,225,539; 5,585,089; 5,693,761; 5,693,762; 5,859,205; It is incorporated herein by reference. In one example, residues in the framework regions of human immunoglobulin one or more variable regions are replaced by corresponding non-human residues (eg, US Pat. Nos. 5,585,089; 5,693,761; 5,693,762; and 6,180,370). In addition, a humanized antibody may comprise residues that are not found in the recipient antibody or the donor antibody. Such alterations can further improve antibody performance (eg, obtaining the desired affinity). In general, an ergonomic antibody will comprise substantially all of at least one, and generally two, variable domains, all and all or all of the hypervariable regions corresponding to that of all or substantially non-human immunoglobulins, or Substantially all of the framework regions are of human immunoglobulin sequence. Ergonomic antibodies optionally comprise at least a portion of an immunoglobulin constant region (Fc), generally a human immunoglobulin constant region. For further details see Jones et al. (1986) Nature 331: 522-525; Riechmann et al. (1988) Nature 332: 323-329; And Presta (1992) Curr. Op. Struct. Biol. 2: 593-596; It is incorporated herein by reference. Thus, such “humanized” antibodies may include antibodies wherein the intact human variable domains are substantially less substituted than those substituted by the corresponding sequence of the non-human species. In practice, the conformable antibody is generally a human antibody in which some CDR residues and possibly some framework residues are substituted by residues of homologous sites in rodent antibodies. For example, US Pat. No. 5,225,539; 5,585,089; 5,693,761; 5,693,762; See 5,859,205. See also US Pat. No. 6,180,370, and International Publication No. WO 01/27160, which discloses a method for producing a humanized antibody and a humanized antibody having improved affinity for a predetermined antigen.

Humanized anti-CD40 antibodies can be produced using Human Engineering ™ technology (Xoma Ltd., Berkeley, California).

Ergonomic anti-CD40 monoclonal antibodies include antibodies such as SGN-40 (Tai et al. (2004) Cancer Res. 64: 2846-52; US Pat. No. 6,838,261), which includes murine anti-CD40 antibody SGN- A humanized form of 14 (Francisco et al. (2000) Cancer Res. 60: 3225-31), and the antibody disclosed in US Patent Application No. 2004/0120948; It is hereby incorporated by reference in its entirety.

The present invention may also be carried out using xenogeneic or modified antibodies produced in non-human mammalian hosts characterized by activating endogenous immunoglobulin (Ig) loci, more particularly transgenic mice. In such transgenic animals, endogenous gene components for expression of the light and heavy chain subunits of the host immunoglobulin confer non-functionality and are replaced by homologous human immunoglobulin loci. Such transgenic animals produce human antibodies in the substantial absence of light or heavy chain host immunoglobulin subunits. For example, US Pat. Nos. 5,877,397 and 5,939,598, which are incorporated herein by reference.

Thus, in one embodiment of the present invention, the whole human antibody of CD40 can be obtained, for example, by immunizing transgenic mice. Such mice can be obtained using XenoMouse® technology (Abgenix; Fremont, California), which is disclosed in US Pat. Nos. 6,075,181, 6,091,001, and 6,114,598, all of which are incorporated herein by reference. For example, to produce CHIR-12.12 antibodies, transgenic mice of human IgG ₁ heavy chain locus and human κ light chain locus are immunized with Sf9 cell expressing human CD40. Mice can also be transgenic from other isotypes. Whole human anti-CD40 antibodies useful in the methods of the invention have been characterized by binding properties similar to those exerted by CHIR-12.12 monoclonal antibodies.

As mentioned above, the term “antibody” as used herein encompasses antibody fragments capable of binding antigen. "Antibody fragments" include portions of intact antibodies, preferably antigen-binding or variable regions of intact antibodies. Examples of antibody fragments include Fab, Fab, F (ab ') 2, and Fv fragments; Dimers; Linear antibodies (Zapata et al. (1995) Protein Eng. 10: 1057-1062); Single-chain antibody molecules; And multispecific antibodies formed from antibody fragments. Papain digestion of the antibody produces two identical antigen-binding fragments called "Fab" fragments, each with a single antigen-binding site and residue "Fc" fragments, whose names are their easy Reflects crystallization ability Pepsin treatment yields F (ab ') 2 fragments at both antigen-binding sites, which allow cross-linking of antigens.

"Fv" is the minimum antibody fragment which contains a complete antigen recognition and binding site. This region consists of a dimer of a solid, non-covalent bond of one heavy chain and one light chain variable domain. It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of the Fv containing only antigen specific 3 CDRs) retains antigen recognition and binding capacity even with lower affinity than the entire binding site.

The Fab fragment contains the constant domain of the light chain and the first constant domain of the heavy chain (C _H 1). Fab fragments differ from Fab 'fragments by the addition of minor residues at the carboxy terminus of the heavy chain C _H 1 domain comprising one or more cysteines of the antibody hinge region. Fab'-SH is assigned herein Fab 'and the cysteine residue (s) of the constant domains contain free thiol groups. Fab 'fragments are produced by reducing the heavy chain disulfide bridge of the F (ab') 2 fragment. Other chemical couplings of antibody fragments are already known.

Fragments of anti-CD40 antibodies are suitable for use in the methods of the present invention as long as they retain the desired affinity of the full length antibody. Thus, for example, fragments of the anti-CD40 antibody retain the ability to bind to the CD40 antigen. Such fragments are characterized by properties similar to the corresponding full length antibodies. Thus, for example, fragments of full-length antagonist anti-CD40 antibodies may be specifically bound to human CD40 antigens, preferably expressed on the surface of human cells, and when binding to CD40 antigens on human CD40-expressing cells, There is no significant agonist activity but antagonist activity. Such fragments are referred to herein as "antigen-binding" fragments. Fragments of anti-CD40 antibodies used in the methods of the invention preferably retain the ability to bind to the appropriate FcR or FcR. Thus, for example, fragments of anti-CD40 antibodies may retain the ability to bind FcγRIIIa. Thus, for example, fragments of full-length anti-CD40 antibodies are capable of specific binding to cell-surface CD40 antigens and binding to FcγRIIIa on human effector cells, such as natural killer (NK) cells. Such fragments are referred to herein as "FcR-binding" fragments. Such fragments generally comprise at least a portion of the constant domain of the heavy chain.

Various techniques have been developed for the production of antibody fragments. Traditionally, such fragments have been derived through proteolysis of intact antibodies (eg, Morimoto et al. (1992) Journal of Biochemical and Biophysical Methods 24: 107-117 (1992) and Brennan et al. (1985) Science 229 : 81). However, such fragments can now be produced directly by recombinant host cells. For example, antibody fragments can be isolated from the antibody phage libraries described above. In other embodiments, the Fab'-SH fragment is E. coli. It can be recovered directly from E. coli and can be chemically coupled to form F (ab ') 2 fragments (Carter et al. (1992) Bio / Technology 10: 163-167). According to another method, F (ab ') 2 fragments can be isolated directly from recombinant host cell culture. Other techniques for producing antibody fragments are apparent to those skilled in the art.

Suitable antigen-binding fragments of antibodies include portions of full-length antibodies, generally antigen-binding or variable regions thereof. Examples of antibody fragments include, but are not limited to, Fab, F (ab ') ₂ , and Fv fragments and single-chain antibody molecules. By monovalent antigen-binding fragments of immunoglobulins consisting of parts of the light and heavy chains. F (ab ') ₂ means a bivalent antigen-binding fragment of an immunoglobulin containing two light chains and part of both heavy chains. A "single-chain Fv" or "sFv" antibody fragment refers to a fragment comprising the VH and VL domains of an antibody, which domain is present in a single polypeptide chain. See, eg, US Pat. Nos. 4,946,778, 5,260,203, 5,455,030, and 5,856,456, which are incorporated herein by reference. In general, the Fv polypeptide further binds with a polypeptide linker between the VH and VL domains which allows the sFv to form the desired structure for antigen binding. For the study of sFv, Pluckthun (1994) The Pharmacology of Monoclonal Antibodies, Vol. 113, ed. Rosenburg and Moore (Springer-Verlag, New York), pp. See 269-315. Antigen-binding fragments of the antagonist anti-CD40 antibodies disclosed herein may be conjugated to a cytotoxin, affecting the killing of target cancer cells, as described below.

In one embodiment of the invention, the anti-CD40 antibody is an antagonist anti-CD40 antibody. If such antibodies bind to CD40 expressed on the surface of human cells, such as human B cells, they do not cause significant agonist activity. In one embodiment of the invention, they bind to CD40 expressed on the surface of human cells to inhibit proliferation and differentiation of such human cells. Anti-CD40 antibodies suitable for use in the methods of the invention include such antibodies that can exhibit antagonist activity against normal and malignant human cells expressing cell-surface CD40 antigens.

"Agonist activity" means a substance that functions as an agent. The agent binds to a receptor on the cell and initiates a reaction or activity similar or identical to that initiated by the natural ligand of the receptor. Agents of CD40 induce any or all of the following responses: B cell proliferation and / or differentiation; Upregulation of intercellular binding via such molecules, ICAM-I, E-selectin, VCAM, and the like; Secretion of pre-inflammatory cytokines such as IL-I, IL-6, IL-8, IL-12, TNF, and the like; Pathways such as TRAF (eg TRAF2 and / or TRAF3), MAP kinases such as NIK (NF-κB including kinases), I-kappa B kinases (IKK α / β), transcription factors NF-κB, Ras and Signaling through the CD40 receptor by the MEK / ERK pathway, PI3K / AKT pathway, P38 MAPK pathway, and the like; Molecules such as XIAP, mcl-1, bcl-x, and the like; B and / or T cell memory generation; B cell antibody production; Up-regulation of B cell isotype switching, cell-surface expression of MHC class II and CD80 / 86.

By "significant" agonist activity is at least 30%, 35%, 40%, 45%, 50%, 60%, 70% of agonist activity over that of a neutral or negative control as measured by a measure of B cell response. 75%, 80%, 85%, 90%, 95%, or 100% larger. Preferably, "significant" agonist activity refers to at least 2-fold or at least 3-fold greater agonist activity than that of a neutral substance or a negative control as measured by a measure of B cell response. Thus, for example, if the subject B cell response is B cell proliferation, the "significant" agonist activity is at least 2-fold greater or at least 3-fold greater than the level of B cell proliferation induced by the neutral or negative control. Induces B cell proliferation levels. In one embodiment of the invention, non-specific immunoglobulins, such as IgG1 that do not bind CD40, serve as negative controls. A substance without "significant agonist activity" exhibits at least about 25% more agonist activity than a natural or negative control-induced agonist activity, and agonist activity by neutral or negative controls as measured by the measurement of B cell responses. More preferably at least about 20%, at least 15%, at least 10%, at least 5%, at least 1%, at least 0.5%, or at least about 0.1% of agonist activity.

By "antagonist activity" is meant a substance that functions as an antagonist. Antagonists of CD40 block or reduce the induction of all responses induced by the binding of CD40 receptors to agonist ligands, in particular CD40L. Antagonists may induce the induction of all one or more agent binding reactions by 5%, 10%, 15%, 20%, 25%, 30%, 35%, preferably 40%, 45%, 50%, 55%, 60 %, More preferably 70%, 80%, 85%, and most preferably 90%, 95%, 99%, or 100%. Determination of CD40 ligand binding specificity and antagonist activity of anti-CD40 therapeutic agents, such as anti-CD40 antibodies, is known, including standard competitive binding assays, immunoglobulin secretion monitoring assays of B cells, B cell proliferation assays, and half body ( Banchereau) -like-B cell proliferation assay, T cell helper assay for antibody production, adjuvant stimulus measurement of B cell proliferation, and assays for up-regulation of B cell activation markers. See, for example, the assays disclosed in WO 00/75348 and US Pat. No. 6,087,329, which is incorporated herein by reference. See also WO 2005/044854, WO 2005/044304, WO 2005/044305, WO 2005/044306, WO 2005/044855, WO 2005/044307, and WO 2005 / 044294WO, the contents of which are incorporated herein by reference. It is included as a reference.

Antagonist / agonist activity deficiency can be assessed by assay, and CHIR-12.12 shows a deficiency of agonist activity. Suitable measurements are shown in the measurements described in US Pat. No. 5,677,165 (Chiron Corporation).

In one embodiment of the invention, the antagonist anti-CD40 antibody lacks significant agonist activity in one cell response. In another embodiment of the invention, the antagonist anti-CD40 antibody has no significant agonist activity as measured (eg proliferation and differentiation, or proliferation, differentiation, and antibody production in B cells).

Of particular interest are antagonist anti-CD40 antibodies which bind to CD40 antigen to human B cells but do not have significant agonist activity as defined herein but exhibit antagonist activity. In one embodiment of the invention, the antagonist anti-CD40 antibody lacks significant agonist activity in the B cell response. In another embodiment of the invention, the antagonist anti-CD40 antibody lacks significant agonist activity as measured in one or more B cell responses (eg, proliferation and differentiation, or proliferation, differentiation, and antibody production).

All known assays can be used to determine whether an anti-CD40 antibody acts as an antagonist of one or more B cell responses. In one embodiment of the invention, the anti-CD40 antibody is characterized by B cell proliferation, B cell differentiation, antibody production, intercellular binding, B cell memory generation, isotype switching, cell-surface expression of MHC class II and CD80 / 86. Acts as an antagonist of at least one B cell response selected from the group consisting of up-regulation, and secretion of pre-inflammatory cytokines such as IL-8, IL-12, and TNF. Of particular interest are antagonist anti-CD40 antibodies that do not have significant agonist activity with respect to B cell proliferation when the human CD40 antigen binds to the surface of human B cells.

The anti-CD40 antibody may be an antagonist of B cell proliferation induced by soluble or cell-surface CD40L as measured in B cell proliferation assays. Suitable B cell proliferation assays are known in the art. Suitable B cell proliferation assays are described below. In one embodiment of the invention, the antagonist anti-CD40 antibody stimulates B cell proliferation at a level of at least about 25% more than B cell proliferation induced by natural substances or negative controls, and is induced by natural substances or negative controls More preferably, at least about 20%, at least 15%, at least 10%, at least 5%, at least 1%, at least 0.5%, or at least about 0.1% or more of B cell proliferation.

In another embodiment of the invention, the anti-CD40 antibody is an antagonist of B cell proliferation induced by another anti-CD40 antibody, eg, an S2C6 anti-CD40 antibody as measured in B cell proliferation, The level of B cell proliferation stimulated by other anti-CD40 antibodies in the presence of -CD40 antibody is at least about 25% (ie, at least) of B cell proliferation induced by other anti-CD40 antibodies in the absence of antagonist anti-CD40 antibodies. 75% inhibition), preferably at least about 20%, 15%, 10%, 5%, 1%, 0.5% of B cell proliferation induced by another anti-CD40 antibody in the absence of antagonist anti-CD40 antibody, Or at least about 0.1%.

In another embodiment of the present invention, the anti-CD40 antibody is an antagonist of B cell proliferation induced by cell line EL4B5, as measured by B cell activation assay, and by EL4B5 cell line in the presence of antagonist anti-CD40 antibody. The level of stimulated B cell proliferation is at least about 25% (ie at least 75% inhibition) of B cell proliferation thereby induced in the absence of cell line antagonist anti-CD40 antibody, preferably in the absence of cell line antagonist anti-CD40 antibody At least about 20%, 15%, 10%, 5%, 1%, 0.5%, or at least about 0.1% of B cell proliferation thereby induced.

In another embodiment of the invention, the anti-CD40 antibody is an antagonist of human T-cell-induced antibody production by human B cells as measured by human T-cell helper assay for antibody production by B cells. . In this manner, the level of IgG antibody production, IgM antibody production, or both IgG and IgM antibody production by B cells stimulated by T cells in the presence of antagonist anti-CD40 antibody is in the absence of antagonist anti-CD40 antibody. Individual antibodies by B cells stimulated by T cells in the absence of at least about 50% (ie at least 75% inhibition) of individual antibody production by B cells stimulated by T cells, preferably in the absence of antagonist anti-CD40 antibodies At least about 25%, 20%, 15%, 10%, 5%, 1%, 0.5%, or at least about 0.1% of production. Additional antagonist anti-CD40 antibodies include monoclonal antibodies referred to as 5D12, 3A8 and 3C6, which are secreted by hybridomas having ATCC grant numbers HB 11339, HB 12024 and HB 11340, respectively. See, for example, US Pat. No. 6,315,998, which is hereby incorporated by reference.

For example, the following assays can be used to assess antagonist activity of anti-CD40 antibodies. Human B cells for this assay can be obtained, for example, from the tonsils obtained through tonsillectomy from an individual, which is described in De Groot et al. (1990) Lymphokine Research (1990) 9: 321. Briefly, tissue is dispersed by a surgical blade, phagocytes and NK cells are removed by treatment with 5 mM L-leucine methyl ester, and T cells are treated with 2-aminoethyl isothiouronium bromide (sheep). ) Removed by one cycle of rosetting red blood cells (SRBC). The purity of the resulting B lymphocyte preparation was FITC-conjugate F of anti- (CD20) mAbBl (Coulter Clone, Hialeah, FA) or anti- (CD3) mAb OKT3 (Ortho, Raritan, NJ) and rabbit anti- (mouse Ig). (ab ') ₂ sections (Zymed, San Francisco, Calif.) were examined by indirect immunofluorescent labeling, and FACS analysis.

B-cell proliferation assay

B cells (4 × 10 ⁴ / well) are incubated with 200 μl IMDM supplemented with 10% fetal calf serum in flat lower 96-well microtiter plates. B cells are stimulated by the addition of immobilized anti- (IgM) antibodies (immunological beads; 5 μg / ml; BioRad, Richmond, California). If desired, 100 U / ml recombinant IL-2 is added. Test monoclonal antibody (mAb) is added at various concentrations at the start of microculture and growth is assessed by measuring the incorporation of ( ³ H) -thymidine for 3 days at 18 hour intervals. Antagonist anti-CD40 antibodies do not significantly adjuvant human B-cell proliferation in the presence of immobilized anti-IgM or immobilized anti-IgM and IL-2.

In half ( Banchereau ) -Like B-cell proliferation assay

Banchereau et al. (1991) Mouse 3T6 transfected cells expressing the HR allele of human FcγRII to test the ability of anti-CD40 monoclonal antibodies to stimulate B-cell proliferation in culture systems similar to those described in Science (1991) 251: 70 Was used. B cells (2 × 10 ⁴ / well) were treated with 200 μl IMDM and 100 U / ml recombinant IL-4 supplemented with 10% fetal calf serum in the presence of 1 × 10 ⁴ transfected cells (irradiated at 5000 Rad). Were incubated in planar-bottom microwells. Prior to B cell addition, 3T6 cells are allowed to adhere to the culture plastics for at least 5 hours. Anti-CD40 mAb is added at various concentrations from 15 ng / ml to 2000 ng / ml, and proliferation of B cells is assessed by measuring thymidine incorporation with [ ³ H] thymidine for 7 days at 18 hour intervals.

Antagonist Anti- CD40 mAb Using S2C6 Inhibition of stimulatory B-cell proliferation

Antagonist anti-CD40 monoclonal antibody (mAb) is an anti-CD40 antibody such as S2C6 (also known as SGN-14, reported as an agent of CD40 stimulation of normal B cell proliferation, using the B-cell proliferation assay described above; Francisco et al. al. (2000) Cancer Res. 60: 3225-3231). Human amygdala B cells (4 × 10 ⁴ / well) were in 200 μl microwells in the presence of anti-IgM bound with Sepharose beads (5 μg / ml) and anti-CD40 mAb S2C6 (1.25 μg / ml) Incubated. Subject anti-CD40 mAb is added at various concentrations and [ ³ H] -thymidine incorporation is assessed after 3 days. As a control, anti- (glucocerebrosidase) mAb 8E4 can be added at a similar concentration. Barneveld et al. (1983) Eur. J. Biochem. See 134: 585. Antagonist anti-CD40 antibodies can inhibit, for example, at least 75% or more of co-stimulation of anti-IgM induced human B-cell proliferation by mAb S2C6 (ie, in the presence of antagonist anti-CD40 antibodies S2C6-stimulated proliferation was only 25% of the thickening observed in the absence of antagonist anti-CD40 antibody). In contrast, no significant inhibition was observed with an equivalent amount of irrelevant mAb 8E4 modulating β-glucocerebrosidase. Barneveld et al., Formerly. These results indicate that anti-CD40 mAb does not deliver a stimulus signal for the proliferation of human B cells, but on the contrary, may inhibit the stimulus signal generated by triggering CD40 with another mAb.

EL4B5  B-cell activation assay by cells

Zubler et al. (1985) J Immunol. (1985) At 134: 3662, the mutant subclones of the mouse thymus EL-4 cell line (EL4B5) strongly stimulate the B cell replication origin of both murine and human to proliferate and differentiate into immunoglobulin-secreting plasma cells in vitro. Observed the fact that it can be made. This activation is antigen-independent and is known to have no MHC restriction. For optimal stimulation of human B cells, the presence of supernatant of activated human T cells is required and when EL4B5 cells are pre-reacted with phorbol-12-myritate 13-acetate (PMA) or IL-1, B-cell responses also occur. Zubler et al. (1987) Immunological Reviews 99: 281; And Zhang et al. (1990) J Immunol. 144: 2955. B-cell activation in this culture system is efficient—limiting dilution experiments have shown that the majority of human B cells can be activated to proliferate and differentiate into antibody-secreting cells. Wen et al. (1987) Eur. J Immunol. 17: 887.

B cells (1000 / well) were treated with 10% heat-inactivated fetal bovine serum, 5 ng / ml phorbol-12-myrilate, with irradiated (5000 Rad) EL4B5 cells (5 × 10 ⁴ / well). Cultured in flat bottom microtiter plates with 200 μl IMDM supplemented with 13-acetate (Sigma) and 5% human T-cell supernatant. Addition of mAb at the time of culture initiation at various concentrations and, ^[3 H] - thymidine incorporation was assessed the in 6 days 18 time intervals thymidine. For the preparation of T-cell supernatants, purified T cells were incubated for 36 hours at a concentration of 10 ⁶ / ml in the presence of 1 μg / ml PHA and 10 ng / ml PMA. Wen et al. (1987) Eur. J. Immunol (1987) 17: 887. T-cell supernatants are obtained by cell centrifugation and stored at -20 ° C. The effect of T-cell supernatants promoting the proliferation of human B cells in EL4B5-B cell culture was tested and the most effective supernatants were collected for use in the experiment. When assessing the effect of anti-CD40 antibody on EL4B5-induced human B-cell proliferation, as a control, a monoclonal antibody such as MOPC-141 (IgG2b) can be added.

Antagonist anti-CD40 antibodies can inhibit, for example, at least 75% or more, B-cell proliferation stimulated by EL4B5 cell lines (ie, EL4B5-induced B cell proliferation in the presence of antagonist anti-CD40 antibodies). Is only 25% of the proliferation observed in the absence of antagonist anti-CD40 antibody). In contrast, control antibodies such as MOPC-141 have no significant effect on EL4B5-induced B cell proliferation.

Human T for Antibody Production by B Cells cell helper  Measure

Antagonist Anti-CD40 antibodies can function as antagonists of immunoglobulin production by B cells. Anti-CD40 antibodies can be tested for this type of antagonist activity by assessing the inhibitory ability of antibodies of immunoglobulin production by B cells with stimulating T cell contact-dependent mode in T cell helper assays. In this manner, 96-well tissue culture plates were coated with a 1: 500 dilution of anti-CD3 mAb CLB-T3 / 3 (CLB, Amsterdam, The Netherlands) plural fluids. As indicated, costimulatory mAbs were added: anti-CD2 mAb CLB-Tl 1.1 / 1 and CLB-Tl 1.2 / 1 (CLB, Amsterdam, The Netherlands), ascites 1: 1000 and anti-CD28 mAb CLB-28 / 1 All (CLB, Amsterdam, The Netherlands). Then, tonsil T cells (irradiation, 3000 Rad; 10 ⁵ / well), tonsil B cells (104 / well), and rIL-2 (20 U / ml) were added. The final volume of each cell culture is 200 μl. After 8 days, cells were centrifuged to harvest the cell free supernatant. The concentrations of human IgM and IgG in the sample (diluted) were measured by the following ELISA.

In one embodiment of the invention, human amygdala B cells (10 ⁴ / well) are coated with anti-CD3 mAb, or irradiated with purified T cells (3000 rad, 10 ⁵ / well) irradiated, or T The cells were cultured in 96-well plates coated with or without other mAbs for costimulation of the cells. After 8 days of culture, supernatants were harvested to measure immunoglobulin production by B cells. Immunoglobulin production by B cells was assessed by the following ELISA assay. Subject anti-CD40 antibodies were added at various concentrations at the start of the culture. As a control, mAb MOPC-141 was added.

Antagonist anti-CD40 antibodies can inhibit at least 50% or more of IgG and IgM antibody production of B cells stimulated by human T cells (ie, T cells by B cells in the presence of antagonist anti-CD40 antibodies- Inducible antibody production was only 50% of the antibody production observed in the absence of antagonist anti-CD40 antibody). In contrast, control antibodies such as MOPC-141 have no significant effect on T cell-induced antibody production by B cells.

Immunoglobulins  For quantification ELISA  Measure

 The concentrations of human IgM and IgG were measured by ELISA. 96-well ELISA plates were prepared with 0.05 M carbonate of 4 μg / ml mouse anti-human IgG mAb MH 16-01 (CLB3 Amsterdam, The Netherlands) or 1.2 μg / ml mouse anti-human IgM mAb 4102 (Tago, Burlingame, CA). Covered with buffer (pH = 9.6) and incubated at 4 ° C. for 16 hours. Plates were washed three times with PBS-0.05% Tween-20 (PBS-Tween) and saturated with BSA for 1 hour. After washing twice, the plates were incubated at 37 ° C. for 1 hour with different dilutions of the test sample. After three washes, bound Ig was 37 ° C. for 1 hour with 1 μg / ml peroxidase-labeled mouse anti-human IgG mAb MH 16-01 (CLB) or mouse anti-human IgM mAb MH 15-01 (CLB). It is detected by culturing. The plate is washed four times and bound peroxidase activity is shown by the addition of O-phenylenediamine as substrate. Human standard serum (H00, CLB) was used to establish a standard curve for each measurement.

Antagonist anti-CD40 antibodies are known in the art. For example, human anti-CD40 antibodies produced by hybridomas designated F4-465 are disclosed in US Patent Applications Nos. 20020142358 and 20030059427; It is incorporated herein by reference with integrity. F4-465 was obtained from HAC mice (Kuroiwa et al. (2000) Nature Biotech 10: 1086 (2000)) and thus expresses human lambda light chains. See also WO 2005/044854, WO 2005/044304, WO 2005/044305, WO 2005/044306, WO 2005/044855, WO 2005/044307, and WO 2005/044294 WO, each of which is incorporated herein by reference. It is included as a reference.

In addition to antagonist activity, the anti-CD40 antibodies used in the methods of the present invention will preferably retain the mechanism of activity against another target cell. For example, the anti-CD40 antibody preferably retains ADCC activity. In other embodiments, the variable region of the anti-CD40 antibody may be expressed on another antibody isotype that retains ADCC activity. It may be conjugated with antigen-binding fragments, therapeutic agents, or radioactive metal ions or radioisotopes of an anti-CD40 antibody against natural, recombinant, or cytotoxin, as further described herein.

As described elsewhere herein, when compared to other antibodies, the inventors found that anti-CD40 antibodies, such as CHIR-12.12, are two FcγRIIIa amino acid 158 allotypes (V or F) against natural killer (NK) cells in human patients. It has been surprisingly found that binding to one can mediate potential antibody-dependent cellular cytotoxicity (ADCC) of CD40-expressing target cells. Thus, anti-CD40 antibodies such as CHIR-12.12 are heterozygous or homozygous for FcγRIIIa-158F (genotype V / F or F / F), and homozygous for FcγRIIIa-158V (genotype V / V) ) Can be used for the treatment of cancer and precancerous conditions associated with CD40-expressing cells of human patients. The present invention is particularly advantageous in the treatment of cancer and precancerous conditions that do not respond to the treatment of Rituximab (Rituxan®), indicating that the clinical activity of Rituximab in NHL correlates with the FcγRIIIa genotype of the patient. Because I bet.

Thus, particularly preferred anti-CD40 antibodies used in the methods of the invention mediate ADCC of CD40-expressing cells by natural killer cells (NK cells) expressing human effector cells, such as FcγRIIIa, with antagonist activity. It can be. Most preferred are anti-CD40 antibodies capable of binding to both FcγRlIIa-158F and FcγRIIIa-158V with high affinity, as further described herein.

Particularly preferred anti-CD40 antibodies are those disclosed in WO 2005/044854, WO 2005/044304, WO 2005/044305, WO 2005/044306, WO 2005/044855, WO 2005/044307, and WO 2005/044294 WO. Is incorporated herein by reference in its entirety.

In particular the subject matter of the CHIR-12.12 monoclonal antibodies described in WO 2005/044854, WO 2005/044304, WO 2005/044305, WO 2005/044306, WO 2005/044855, WO 2005/044307, and WO 2005/044294. Antagonist anti-CD40 antibody that shares binding properties. Such antibodies include, but are not limited to:

a) monoclonal antibody CHIR-12.12;

b) monoclonal antibodies produced by hybridoma cell line 12.12;

c) the sequence shown in SEQ ID NO: 2, the sequence shown in SEQ ID NO: 4, the sequence shown in SEQ ID NO: 5, both sequences shown in SEQ ID NO: 2 and SEQ ID NO: 4, and SEQ ID NO Monoclonal antibodies comprising an amino acid sequence selected from the group consisting of: 2 and both sequences shown in SEQ ID NO: 5;

d) a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of a sequence shown in SEQ ID NO: 1, a sequence shown in SEQ ID NO: 3, and both sequences shown in SEQ ID NO: 1 and SEQ ID NO: 3 Monoclonal antibodies having an amino acid sequence encoded by;

e) monoclonal antibodies that bind to epitopes capable of binding monoclonal antibodies produced by the binding hybridoma cell line 12.12;

f) a monoclonal antibody that binds to an epitope comprising residues 82-87 of the human CD40 sequence shown in SEQ ID NO: 7 or SEQ ID NO: 9;

g) a monoclonal antibody that binds to an epitope comprising residues 82-89 of the human CD40 sequence shown in SEQ ID NO: 7 or SEQ ID NO: 9;

h) monoclonal antibodies that compete with monoclonal antibody CHIR-12.12 in a competitive binding assay;

i) a monoclonal antibody of item a) above or a monoclonal antibody of any one of items c) -h) above, wherein said antibody is recombinantly produced; And

j) A monoclonal antibody, which is an antigen-binding fragment of the monoclonal antibody of any one of the preceding items a) -i), wherein said fragment retains the ability to specifically bind a human CD40 antigen.

Monoclonal antibody CHIR-12.12 is particularly preferred for use in the methods of the invention.

The monoclonal antibody CHIR-12.12 has been described in detail in WO 2005/044854, WO 2005/044304, WO 2005/044305, WO 2005/044306, WO 2005/044855, WO 2005/044307, and WO 2005/044294. The CHIR-12.12 antibody is a whole human anti-CD40 monoclonal antibody of the IgG1 isotype produced from the hybridoma cell line 153.8E2.D10.D6.12.12 (cell line 12.12). Cell lines were generated using splenocytes of immunized heterologous mice (XenoMouse® technology; Abgenix; Fremont, California) containing human IgG1 heavy chain loci and human κ chain loci. Interest cells were fused with mouse myeloma SP2 / 0 cells (Sierra Biosource). Results The hybridomas were sub-cloned several times to produce a stable monoclonal cell line 12.12. Other antibodies suitable for use in the methods of the present invention could be similarly prepared using mouse transgenic for human immunoglobulin loci, as described herein.

The CHIR-12.12 monoclonal antibody binds to soluble CD40 in ELISA-type assays and blocks binding of CD40-ligands to cell-surface CD40, displacing pre-binding CD40-ligands as determined by flow cytometry. Antibodies CHIR-5.9 and CHIR-12.12 compete with each other to bind CD40 but do not compete with 15B8, an anti-CD40 monoclonal antibody, which is described in US Provisional Application No. 60 / 237,556, “Human Anti-CD40 Antibody,” Octocer. 2, 2000,) and PCT International Application PCT / US01 / 30857 “Human Anti-CD40 Antibody” (October 2, 2001) (agent designation PP 16092.003) and WO 2002/028904, which are integral and It is included as a reference. For in vitro tests on the effect of B cell proliferation in normal human subjects, CHIR-12.12 acts as an antagonist anti-CD40 antibody. In addition, CHIR-12.12 does not induce strong proliferation of human lymphocytes in normal subjects. Antibodies can kill CD40-expressing target cells by antibody dependent cytotoxicity (ADCC). The binding affinity of CHIR-12.12 for human CD40 is 5 × 10 ⁻¹⁰ M as measured by the Biacore ™ assay.

Nucleotide and amino acid sequences of the variable regions of the CHIR-12.12 antibody are provided herein. More specifically, the amino acid sequences for the leader, variable, and constant regions of the light and heavy chains for mAb CHIR-12.12 are shown in SEQ ID NO: 2 (complete sequence for the light chain of mAb CHIR-12.12), SEQ ID NO: 4 (complete sequence for heavy chain of mAb CHIR-12.12), and SEQ ID NO: 5 (complete sequence or variant of heavy chain of mAb CHIR-12.12 as shown in SEQ ID NO: 4, wherein the variant is SEQ ID NO: 4 And a serine substitution of an alanine residue at position 153). The nucleotide sequences encoding the light and heavy chains of mAb CHIR-12.12 are shown in SEQ ID NO: 1 (light chain coding sequence for mAb CHIR-12.12) and SEQ ID NO: 3 (heavy chain coding sequence for mAb CHIR-12.12). . Hybridomas expressing the CHIR-12.12 antibody were deposited with ATCC under Patent Accession No. PTA-5543.

Anti-CD40 antibodies used in the methods of the present invention are antibodies that differ from CHIR-12.12 monoclonal antibodies but that contain CDRs, and that have one or more amino acid addition (s), deletion (s), or substitution (s). Include. Anti-CD40 antibodies used in the methods of the invention may be de-immunized antibodies, in particular de-immunized antagonist anti-CD40 antibodies, as described, for example, in International Publication Nos. WO 98/52976 and WO 0034317 Can be produced together; It is incorporated herein by reference. In this manner, the residues in the antagonist anti-CD40 antibodies of the present invention are modified to confer antibodies with human antagonist activity against human CD40-expressing cells while conferring non-immunogenic or low immunogenicity to humans, Here, this activity is measured by the assay described herein. Also within the scope of the present invention is a fusion protein comprising a subject antibody, eg, an antagonist anti-CD40 antibody or an antagonist anti-CD40L antibody, or fragments thereof, wherein the fusion protein is as known in the art. Can be synthesized or expressed from the corresponding polynucleotide vector. Such fusion proteins are described with regard to the conjugation of antibodies as described herein.

All known antibodies with binding specificity of interest can retain sequence variations produced using the methods described, for example, in Patent Publication Nos. EP 0983303 Al, WO 00/34317, and WO 98/52976. Incorporated herein by reference. For example, Knee shows that sequences in CDRs can allow antibodies to bind to MHC class II and trigger unwanted helper T-cell responses. Conservative substitutions may allow antibodies to retain their binding activity but lose their ability to trigger unwanted T-cell responses. All such conservative or non-conservative substitutions can be made using methods known in the art, such as those mentioned herein, and the resulting antibodies can be used in the methods of the invention. Variant antibodies can typically be tested for specific activity, such as antagonist activity, affinity, and specificity, using the methods described herein.

For example, amino acid sequence variants of antagonist anti-CD40 antibodies, such as CHIR-12.12 monoclonal antibodies, can be prepared by mutations in cloned DNA sequences encoding the antibody of interest. Methods of mutagenesis and nucleotide sequence alteration are well known in the art. For example, Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York); Kunkel (1985) Proc. Natl. Acad. Sci. USA 82: 488-492; Kunkel et al. (1987) Methods Enzymol. 154: 367-382; Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor, New York); US Patent No. 4,873,192; And references cited herein; This is incorporated herein by reference. Guidelines for suitable amino acid substitutions that do not affect the biological activity of the subject polypeptides are described in Dayhoff et al. (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, D. C.), which is incorporated herein by reference. It may be desirable to exchange conservative substitutions, such as one amino acid, with other amino acids having similar properties. Examples of conservative substitutions include, but are not limited to, Gly <=> Ala, Val <=> Ile <=> Leu, Asp <=> Glu, Lys <=> Arg, Asn <=> Gln, and PheoTrp <=> Tyr It is not limited.

In constructing a subject antibody, eg, a subject antagonist anti-CD40 antibody polypeptide variant, the alteration causes the variant to have the desired activity, ie similar binding affinity, and in the case of antagonist anti-CD40 antibodies, Specific binding to the human CD40 antigen expressed on the surface of the cell, and when the CD40 antigen binds on human CD40-expressing cells, exhibits antagonist activity without significant agonist activity. Obviously, all mutations generated in the DNA encoding the variant polypeptides must be located in sequences outside of the reading frame and preferably do not produce complementary regions capable of producing secondary mRNA structures. See EP Patent Application Publication No. 75,444.

In addition, the constant regions of antibodies, eg, antagonist anti-CD40 antibodies, are mutated to alter effector function in a variety of ways. See, eg, US Pat. No. 6,737,056Bl and US Patent Application No. 2004/0132101 Al, which discloses Fc mutations that optimize antibody binding to the Fc receptor.

Preferably, variants of the mentioned antibodies, eg, antagonist anti-CD40 antibodies, are amino acids of the mentioned antibodies, eg, antagonist anti-CD40 antibody molecules, eg, the CHIR-12.12 monoclonal antibodies described herein. Sequence, or at least 70% or 75% sequence match, preferably at least 80% or 85% sequence match, more preferably at least 90%, 91%, 92%, 93%, 94 with a short portion of the mentioned antibody molecule Have an amino acid sequence that matches% or 95% sequence. More preferably, the molecules share at least 96%, 97%, 98% or 99% consensus processing. For purposes of the present invention, the percent sequence agreement is based on the Smith-Waterman homology search algorithm using an affine gap search with a gap open penalty of 12, a gap expansion penalty of 2, and a BLOSUM matrix of 62. Is determined using. Smith-Waterman homology search algorithms are described in Smith and Waterman (1981) Adv. Appl. Math. Can be learned at 2: 482-489. Variants may include, for example, the mentioned antibodies, eg, antagonist anti-CD40 antibodies, less than 1 to 15 amino acid residues, less than 1 to 10 amino acid residues, eg, less than 6-10, 5, 4, Less than 3, 2, or 1 amino acid residue.

With regard to the optimal alignment of two amino acid sequences, contiguous portions of variant amino acid sequences may have additional amino acid residues or deleted amino acid residues relative to the amino acid sequence mentioned. Contiguous portions used to compare with the mentioned amino acid sequences may include at least 20 acid residues and may be 30, 40, 50, or more amino acid residues. Correction of sequence matching related to conservative residue substitutions or gaps can be performed (Smith-Waterman homology search algorithm).

In particular, when the CD40 antigen is bound to malignant B cells, specific binding to CD40 is possible, and the exact chemical structure of the polypeptide having antagonist activity depends on a number of factors. Ionizable amino groups and carboxyl groups are present in the molecule and certain polypeptides can be obtained as acidic or basic salts, or as neutral forms. All such preparations which retain their biological activity when located under suitable environmental conditions are included in the definition of antagonist anti-CD40 antibodies as used herein. In addition, the primary amino acid sequence of a polypeptide can be increased by induction with sugar moieties (glycosylation) or by other supplemental molecules such as lipids, phosphates, acetyl groups and the like. This may be increased by sugars and conjugation. Certain features of this increase can be achieved through post-translational systems of host production; Such other changes may be introduced into the in vitro. In any event, such alterations are included within the definition of anti-CD40 antibodies used herein unless the antagonist properties of the anti-CD40 antibodies are disrupted. It is anticipated that such alterations may affect activity quantitatively or qualitatively by enhancing or decreasing polypeptide activity within various assays. In addition, individual amino acid residues in the chain can be modified by oxidation, reduction, or other induction, and the polypeptide can be degraded to obtain fragments that retain activity. Such alterations that do not destroy antagonist activity do not remove the definition of an anti-CD40 subject antibody used herein in a polypeptide sequence.

This technique provides practical guidance regarding the preparation and use of polypeptide variants. In the production of anti-CD40 antibody variants, one of ordinary skill in the art readily knows which alterations of natural protein nucleotides or amino acid sequences exhibit variations suitable for use as therapeutically active ingredients of the pharmaceutical compositions used in the methods of the present invention. You can decide.

Anti-CD40 antibodies for use in the methods of the present invention preferably retain at least one in vitro and / or in vivo biological activity of: secretion of immunoglobulin secretion by normal human peripheral B cells stimulated by T cells control; Inhibition of survival and / or proliferation of normal human peripheral B cells stimulated by CD40L-expressing cells or soluble CD40 ligands (sCD40L); Inhibition of survival and / or proliferation of normal human peripheral B cells stimulated by Jurkat T cells; inhibition of "survival" anti-apoptotic intracellular signals in all cells stimulated by sCD40L or solid-phase CD40L; And ligation by sCD40L or solid-phase CD40L, deletion of a cell containing cognate ligands of CD40, including but not limited to CD40-containing target cells or T cells and B cells, non-responsive and / or inducing immune tolerance, Induction of expansion or activation of CD4 + CD25 + regulatory T cells (eg, donor homoantigen-specific tissue rejection via the CD40-CD40L interface, van Maurik et al. (2002) J Immunol. 169: 5401-5404), Cytotoxicity through all mechanisms (including but not limited to antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), proliferation in target cells and / or down-regulation of apoptosis), Target cell cytokine secretion and / or regulation of cell surface molecule expression, and inhibition of CD40 signal transduction in all cells with a combination thereof.

Assays of such biological activity may be performed as described herein, and include the provisional application “antagonist anti-CD40 monoclonal antibodies and methods of using them” (2003. 11. 4 / 2003. 11. 26 / and April 27, 2004). ) And US Patent Application Nos. 60 / 517,337 (agent designation number PP20107.001 (035784/258442)), 60 / 525,579 (agent designation number PP20107.002 (035784/271525)), and 60 / 565,710 (agent designation number PP20107). 003 (035784/277214), respectively; And International Patent Application No. PCT / US2004 / 037152 (agent designation number PP20107.004 (035784/282916)) and published in WO 2005/044854, "Agonist anti-CD40 monoclonal antibodies and methods of use thereof" (2004. 11. 4.); The contents of which are hereby incorporated by reference in their entirety. See also Schultze et al. (1998) Proc. Natl. Acad. ScL USA 92: 8200-8204; Denton et al. (1998) Pediatr. Transplant. 2: 6-15; Evans et al. (2000) J. Immunol. 164: 688-697; Noelle (1998) Agents Actions Supp L 49: 17-22; Lederman et al. (1996) Curr. Opin. Hematol. 3: 77-86; Coligan et al. (1991) Current Protocols in Immunology 13:12; Kwekkeboom et al (1993) Immunology 79: 439-444; And the measurements described in US Pat. Nos. 5,674,492 and 5,847,082; This is incorporated herein by reference.

An exemplary assay for detecting antagonist anti-CD40 antibodies specific for the CD40-antigen epitope identified herein is a “competitive binding assay”. Competitive binding assays are serological assays in which unknown antibodies are detected and quantified by the ability to inhibit the binding of known and labeled ligands to their specific antibodies. This is also called a competitive inhibition measure. In a representative competitive binding assay, the labeled CD40 polypeptide is precipitated by the candidate antibody in combination with a monoclonal antibody that occurs against a sample, eg, one or more epitopes of the monoclonal antibodies of the invention. Anti-CD40 antibodies that specifically react with an epitope of interest can be identified by selecting a series of antibodies prepared against a protein fragment comprising a CD40 protein or a specific epitope of the CD40 protein of interest. For example, for human CD40, the subject epitope is shown in SEQ ID NO: 10 and the short isoform (GenBank accession number NM_152854) of human CD40 (GenBank accession number NP_690593) encoded by the sequence shown in SEQ ID NO: 9. Or, linear and / or of human CD40 long isoform (GenBank Accession Nos. CAA43045 and NP_001241, SEQ ID NO: 12, encoded by the sequence shown in SEQ ID NO: 11; GenBank Accession Nos. X60592 and NM_001250) Epitopes comprising non-linear amino acid residues. In other embodiments, competitive binding assays with suitable antagonist anti-CD40 antibodies identified in advance may select monoclonal antibodies that are comparable to previously identified antibodies.

Antibodies used in such immunoassays can be labeled or unlabeled. Unlabeled antibodies can be used for aggregation; Labeled antibodies can be used in a wide variety of assays using a variety of labels. Detection of the formation of the antibody-antigen complex between the anti-CD40 antibody and the subject epitope can be facilitated by adhering the detectable substance to the antibody. Suitable detection means include the use of labels such as radionucleotides, enzymes, coenzymes, fluorescent materials, chemiluminescence, colorants, enzyme substrates or co-factors, enzyme inhibitors, combination complexes, free radicals, particles, pigments, and the like. . Examples of suitable enzymes include carrot peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; Examples of suitable formulator complexes include streptavidin / biotin and avidin / biotin; Examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, monochloro chloride or phycoerythrin; An example of a luminescent material is luminol; Examples of bioluminescent materials include luciferase, luciferin, and aquarin; Examples of suitable radioactive materials are ¹²⁵ I, ¹³¹ I, ³⁵ S, or ³ H. Such labeling reagents can be used in well known assays such as radioimmunoassay, enzyme immunoassay such as ELISA, fluorescence immunoassay, and the like. See, eg, US Pat. No. 3,766,162; 3,791,932; 3,817,837; And 4,233,402.

Antibodies can be genetically engineered to retain increased ADCC activity. In particular, the carboxy-terminal half of the CH2 domain is crucial for ADCC mediated through the FcRIII receptor. Since the CH2 and hinge regions play an important role in effector function, a series of multi-domain antibodies containing extra CH2 and / or hinge regions can be generated and can be investigated for all changes in effector titers ( Greenwood, J., Gorman, SD, Routledge, EG, Lloyd, LS. & Waldmann, H., Ther Immunol. 1994 Oct; l (5): 247-55). Alternative approaches can manipulate extra domains in parallel, for example, to form dimers by manipulating cysteines into the H-chain of chimeric Ig (Shopes B. (1992) J. Immunol. 1992 1; 148 ( 9): 2918-22). In addition, mutations may be introduced into the Fc region (eg US 6,737,056 Bl), cell expression in fucosyl transferase deficient cell lines (eg US2003 / 0115614), or glycosylation of antibodies for changes that increase ADCC activity. It can also be manipulated to influence other changes (eg US Pat. No. 6,602,684).

The present invention is advantageous in the treatment of CD40 expressing cancer and precancerous conditions, wherein the patient is homozygous or heterozygous for the Fcγ RIIIa-158F genotype.

As used herein, "anti-CD20 antibody" encompasses all antibodies that specifically recognize CD20 cell surface antigens and include polyclonal antibodies, monoclonal antibodies, single-chain antibodies, and fragments thereof such as Fab, F ( ab ') 2, Fv, and other fragments retaining the antigen-binding function of the parental anti-CD20 antibody. In particular, subjects of the methods of the invention are anti-CD20 antibodies or antigen-binding fragments thereof that possess the binding properties exhibited by IDEC-C2B8 monoclonal antibodies (Biogen IDEC Inc., Cambridge, Mass.).

In one embodiment of the invention, the anti-CD40 antibody used in the methods of the invention exhibits more potent therapeutic activity than the chimeric anti-CD20 monoclonal antibody IDEC-C2B8, and the anti-tumor activity using human lymphoma or myeloma cell line Equivalent amounts of these antibodies in nude mouse xenograft tumor models are measured. IDEC-C2B8 (IDEC Pharmaceuticals Corp., San Diego, California; available under the trademark Rituxan®, Rituximab) is a murine anti-CD20 monoclonal antibody, IDEC-2B8 (Reff et al. (1994) Blood 83: 435- Chimeric anti-CD20 monoclonal antibody containing human IgG1 and kappa constant regions with murine variable regions isolated from 445). Rituxan® has been approved for the treatment of relapsed B-cell low or vesicle non-Hodgkin's lymphoma (NHL). The discovery of antibodies with superior therapeutic, in particular anti-tumor, activity, compared to Rituxan®, allows for radical improvements in the treatment of cancer and precancerous conditions, such as B cell lymphoma, especially B cell non-Hodgkin's lymphoma. .

Suitable nude mouse xenograft tumor models include the use of human Burkitt's lymphoma cell lines known as Namalwa and Daudi. Preferred embodiments measure anti-tumor activity in an engineered nude mouse xenograft tumor model using the Daudi human lymphoma cell line described in Example 7 herein below. Engineered nude mouse xenograft tumor models using Daudi lymphoma cell lines are more effective than unengineered models in distinguishing the therapeutic efficacy of a given antibody, which is administered only after the tumors have reached a measurable size in engineered model antibodies. This is because it is disclosed. In an unmanipulated model, antibody administration is generally initiated within about 1 day of tumor inoculation and before the tumor can be promoted. The ability of antibodies to exceed Rituxan® (ie, exerted increased therapeutic activity) in engineered models strongly points to the fact that antibodies are therapeutically more effective than Rituxan®. Moreover, in the Daudi model, the target anti-CD20 of Rituxan® is expressed on the cell surface at a higher level than CD40.

An "equivalent amount" of the anti-CD40 antibody and Rituxan® of the present invention means that the same mg dose was administered on a per sugar mass basis. Thus, when the anti-CD40 antibody is administered at 0.01 mg / kg body weight (mouse used in tumor models), Rituxan® is administered at 0.01 mg / kg body weight (mouse). Similarly, when anti-CD40 antibody is administered at 0.1, 1, or 10 mg / kg body weight (mouse used in tumor models), Rituxan® is administered at 0.1, 1, or 10 mg / kg body weight (mouse), respectively. do.

When administered to a nude mouse xenograft tumor model, some anti-CD40 antibodies will result in a significantly reduced tumor volume than equivalent amounts of Rituxan®. For example, when measured in an engineered nude mouse xenograft tumor model using a Daudi human lymphoma cell line in the manner described in Example 7 herein, the whole human monoclonal antibody CHIR-12.12 was observed to be equivalent to that of Rituxan. Compared to at least 20% reduction in anti-tumor activity compared to 50% to 60% increase in anti-tumor activity in this assay. This increased anti-tumor activity reflects a large decrease in tumor volume observed with the anti-CD40 antibodies of the invention when compared to equivalent doses of Rituxan® or when inducing a more complete response. Thus, for example, depending on the length of time after tumor inoculation, the monoclonal antibody CHIR-12.12 exhibits a tumor volume of about 1/3 to about 1/2 of that observed with the equivalent dose of Rituxan®.

Another difference in antibody potency is to determine the in vitro antibody concentration required to obtain maximal lysis of tumor cells in vitro in which NK cells are present. For example, the anti-CD40 antibodies of the invention achieve maximal lysis of Daudi cells at an EC 50 below a Rituxan® concentration of 1/2, and preferably 1/4, and most preferably 1/10. do. This type of measurement has been described in the Examples herein.

 Anti-CD40 antibodies that have the advantage of having significantly greater efficacy than equivalent Rituxan® in the aforementioned assays:

a) monoclonal antibody CHIR-12.12;

b) monoclonal antibodies produced by hybridoma cell line 12.12;

c) the sequence shown in SEQ ID NO: 2, the sequence shown in SEQ ID NO: 4, the sequence shown in SEQ ID NO: 5, both sequences shown in SEQ ID NO: 2 and SEQ ID NO: 4, and SEQ ID NO Monoclonal antibodies comprising an amino acid sequence selected from the group consisting of: 2 and both sequences shown in SEQ ID NO: 5;

d) a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of a sequence shown in SEQ ID NO: 1, a sequence shown in SEQ ID NO: 3, and both sequences shown in SEQ ID NO: 1 and SEQ ID NO: 3 Monoclonal antibodies having an amino acid sequence encoded by;

e) monoclonal antibodies that bind epitopes capable of binding monoclonal antibodies produced by the binding hybridoma cell line 12.12;

f) a monoclonal antibody that binds to an epitope comprising residues 82-87 of the human CD40 sequence shown in SEQ ID NO: 7 or SEQ ID NO: 9;

g) a monoclonal antibody that binds to an epitope comprising residues 82-89 of the human CD40 sequence shown in SEQ ID NO: 7 or SEQ ID NO: 9;

h) monoclonal antibodies that compete with monoclonal antibody CHIR-12.12 in a competitive binding assay;

i) the monoclonal antibody of item a) above or the monoclonal antibody of any one of items c) -h) above, which is recombinantly produced; And

j) A monoclonal antibody which is an antigen-binding fragment of the monoclonal antibody of any one of items a) -i) above, wherein said fragment has the ability to specifically bind a human CD40 antigen.

The present invention provides methods for identifying human patients with cancer or precancerous conditions treatable with an anti-CD40 antibody, including:

a) identifying a human patient with a cancer or precancerous condition associated with CD40-expressing cells; And

b) determining the FcγRIIIa-158 genotype of said human patient (V / V, V / F or F / F);

Wherein the cancer or precancerous condition is treatable with an anti-CD40 antibody when the human patient is heterozygous or homozygous for FcγRIIIa-158F (genotype V / F or F / F). Cancer or precancerous conditions are resistant to the treatment of Rituximab (Rituxan®).

If a human patient with cancer or precancerous condition treatable with an anti-CD40 antibody is identified, then the human patient may be treated with an anti-CD40 antibody. Thus, the method includes (c) administering a therapeutically or prophylactically effective amount of an anti-CD40 antibody to a human patient identified as heterozygous or homozygous for FcγRIIIa-158F (genotype V / F or F / F). It may further comprise.

This method of identifying human patients with cancer or precancerous conditions treatable with an anti-CD40 antibody can be readily performed by those skilled in the art using suitable diagnostic kits. The kit should contain a reagent suitable for determining the FcγRIIIa-158 genotype of a human patient. Accordingly, the present invention also provides kits for identifying human patients with cancer or precancerous conditions treatable with an anti-CD40 antibody, comprising reagents for determining the FcγRIIIa-158 genotype of a human patient. Suitable kits are described in detail elsewhere herein.

The invention also provides a method of selecting antibody therapies for the treatment of a human patient with cancer or precancerous conditions, including:

a) identifying a human patient with a cancer or precancerous condition associated with CD40-expressing cells; And

b) determining the FcγRIIIa-158 genotype of said human patient (V / V, V / F or F / F);

Here, when the human patient is heterozygous or homozygous for FcγRIIIa-158F (genotype V / F or F / F), an anti-CD40 antibody is selected for treatment of the cancer or precancerous condition. The cancer or precancerous condition may be resistant to the treatment of Rituximab (Rituxan®).

If anti-CD40 antibody therapy is selected for the treatment of a human patient with cancer or precancerous condition, then the human patient may be treated with an anti-CD40 antibody. Thus, the method includes the steps of (c) administering a therapeutically or prophylactically effective amount of an anti-CD40 antibody to a human patient identified as heterozygous or homozygous for FcγRIIIa-158F (genotype V / F or F / F). It may further comprise.

This method of selecting antibody therapy for the treatment of human patients with cancer or precancerous conditions can be readily performed by those skilled in the art using suitable diagnostic kits. The kit should contain a reagent suitable for determining the FcγRIIIa-158 genotype of a human patient. Accordingly, the present invention also provides kits for selecting antibody therapy for the treatment of human patients with cancer or precancerous conditions associated with CD40-expressing cells, comprising reagents for determining the FcγRIIIa-158 genotype of a human patient. do.

“Curable with anti-CD40 antibody” means “positive therapeutic response” (herein) with respect to the cancer or precancerous condition to be treated when a human patient (ie, an individual with cancer or precancerous condition) is treated with an anti-CD40 antibody. Definition).

All methods were used to determine the FcγRIIIa-158 genotype of human patients using biological samples obtained from human patients.

For example, the present invention provides a kit for use in determining the FcγRIIIa-158 genotype of a human patient, comprising at least 10 or more nucleotides in length that is suitable for determining the FcγRIIIa-158 genotype of a human patient. A microarray including one probe is included. Labeled RNA or DNA hybridizes with complementary probes on the array and is detected by laser scanning. The hybridization intensity of each probe on the array is measured and converted into quantitative values representing relative gene expression levels. Selection of probe sequence and length can be readily performed by one skilled in the art. The nucleotide sequences of human genes and mRNAs encoding FcγRIIIa-158 F and V allotypes are known. Thus, one of ordinary skill in the art can, under appropriate experimental conditions, select probe (s) that allow for FcγRIIIa-158 genotyping of target sequences.

Synthesis techniques of this arrangement using mechanical synthesis methods are described, for example, in US Pat. No. 5,384,261, which is incorporated herein by reference in its entirety. Although planar array surfaces are preferred, the arrays are fabricated with virtually any surface, or even multiplicity of surfaces. The batch may be a peptide or nucleic acid on beads, gels, polymer surfaces, fibers such as optical fibers, glass or any other suitable substrate, see US Pat. Nos. 5,770,358, 5,789,162, 5,708,153, 6,040,193 and 5,800,992, each of which is integral It is included herein with the last name. The arrangement can be packaged in this manner to allow for the operation of diagnostic or fully-included devices. See, eg, US Pat. Nos. 5,856,174 and 5,922,591, which are incorporated herein by reference.

For example, the present invention also provides a kit used to determine the FcγRIIIa-158 genotype of a human patient, and is used as a primer for polymerase-catalytic amplification of a region of a gene or mRNA encoding amino acid 158 of FcγRIIIa. Suitable oligonucleotides. Selection of primer sequences and lengths can be readily made by those skilled in the art. The nucleotide sequences of human genes and mRNAs encoding FcγRIIIa-158 F and V allotypes are known. Thus, one of ordinary skill in the art can select, under suitable experimental conditions, primers to amplify a region of a gene or mRNA encoding amino acid 158 of FcγRIIIa. The amplified sequence can be sequenced using known methods to determine the FcγRIIIa-158 genotype of a patient.

Another method of determining the FcγRIIIa-158 genotype of a human patient is to use a nucleic acid-based method of detecting DNA fragmentation that is characteristic of the FcγRIIIa-158 genotype of a human patient. When determined using agarose gel electrophoresis, the DNA of each FcγRIIIa-158 genotype has a characteristic pattern. Accordingly, the present invention also provides kits for use in determining the FcγRIIIa-158 genotype of a human patient, comprising one or more restriction enzymes suitable for determining the FcγRIIIa-158 genotype of a human patient. Suitable restriction enzymes are known in the art (eg Koene et al. (1997) Blood 90 (3): 1109-1114).

The kit of the present invention may comprise instructions instructing how to use the kit to determine the FcγRIIIa-158 genotype of a human patient. The kit may also include, for example, a buffer, preservative, or protein stabilizer. Each component of the kit is generally sealed in a separate container, and all the various containers are included in a single package with instructions instructing how to use the kit to determine the FcγRIIIa-158 genotype of a human patient.

The present invention provides the use of an anti-CD40 antibody in the manufacture of a medicament for treating a cancer or precancerous condition associated with a CD40-expressing cell as described herein.

Anti-CD40 antibodies of the invention are administered at therapeutically effective concentrations for the treatment or prevention of cancer or precancerous conditions associated with CD40-expressing cells. To achieve this goal, antibodies can be formulated using various acceptable carriers and / or known excipients. Anti-CD40 antibodies can be administered by administration of the parenteral route. In general, antibodies are administered by infusion intravenously or subcutaneously. Methods of achieving such administration are known to those skilled in the art.

Intravenous administration preferably occurs by infusion over a period of 1 hour up to about 10 hours (1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 hours or less). Subsequent infusions may be administered for a time period of 1 to about 6 hours or less, including, for example, about 1 to about 4 hours, about 1 to about 3 hours, or about 1 to about 2 hours or less. have. In other embodiments, the dosage may be administered subcutaneously.

Pharmaceutical compositions of the invention are formulated to be compatible with the intended route of administration. Solutions or suspensions for parenteral use may include the following components: sterile diluents such as water for injection, saline solution; Antibacterial agents such as benzyl alcohol or methyl parabens; Antioxidants such as ascorbic acid or sodium bisulfite; Chelating agents such as ethylenediaminetetraacetic acid; Buffers such as acetates, citrates or phosphates and isotonicity modifiers such as sodium chloride or dextrose. The pH can be adjusted by acid or base such as hydrochloric acid or sodium hydroxide. Parenteral products may be sealed in ampoules, disposable syringes, or multiple dose vials.

Anti-CD40 antibodies are generally provided by standard techniques, including pharmaceutically acceptable buffers such as sterile saline, sterile buffer, combinations of the foregoing, and the like. Methods for preparing parenterally administrable medicaments are described in Remington's Pharmaceutical Sciences (18th ed .; Mack Publishing Company, Eaton, Pennsylvania, 1990), which is incorporated herein by reference. See also, for example, WO 98/56418, which describes stabilized antibody pharmaceutical formulations suitable for use in the methods of the present invention.

The dosage of at least one anti-CD40 antibody can be readily determined by one skilled in the art. Factors affecting the method of administration and the amount of at least one anti-CD40 antibody are the intensity of the disease, the history of the disease, and the age, height, weight, health, type and physical condition of the subject being treated. Or antibody injection reactions. Similarly, the dosage of the anti-CD40 antibody will depend on the method of administration and whether the subject is single or multiple doses of this antitumor agent. In general, high doses of anti-CD40 antibodies are preferred for weight gain in subjects undergoing therapy.

The single dose of anti-CD40 antibody administered is about 0.3 mg / kg to about 50 mg / kg, about 0.1 mg / kg to about 40 mg / kg, about 0.01 mg / kg to about 30 mg / kg, about 0.1 mg / kg to about 30 mg / kg, about 0.5 mg / kg to about 30 mg / kg, about 1 mg / kg to about 30 mg / kg, about 3 mg / kg to about 30 mg / kg, about 3 mg / kg To about 25 mg / kg, about 3 mg / kg to about 20 mg / kg, about 5 mg / kg to about 15 mg / kg.

Thus, for example, the dosage may be 0.3 mg / kg, 0.5 mg / kg, 1 mg / kg, 1.5 mg / kg, 2 mg / kg, 2.5 mg / kg, 3 mg / kg, 5 mg / kg, 7 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 35 mg / kg, 40 mg / kg, 45 mg / kg, or 50 mg / kg Other dosages will be in the range of about 0.3 mg / kg to about 50 mg / kg.

Treatment of a subject by a therapeutically effective amount of an antibody may comprise a single treatment or, preferably, may comprise a series of treatments. Thus, in another embodiment of the invention, the method may comprise administering multiple doses of the anti-CD40 antibody. This method comprises a pharmaceutical composition comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40 or more anti-CD40 antibodies. Administration of a therapeutically effective dose. Frequency and duration of administration of multiple doses of a pharmaceutical composition comprising an anti-CD40 antibody can be readily determined by one skilled in the art without inappropriate experimentation. The same therapeutically effective dose of anti-CD40 antibody may continue to be administered during the course of treatment. In other embodiments, other therapeutically effective doses of the anti-CD40 antibody may continue to be used during the course of treatment.

In an embodiment, the subject has a body weight of about 0.1-20 mg / kg, once per week for about 1-10 weeks, preferably about 2-8 weeks, more preferably about 3-7 weeks, and more preferably Treated with anti-CD40 antibody in the range of about 4, 5, or 6 weeks. Treatment is carried out every 2 to 12 months to prevent relapse or if there are signs of relapse. It is apparent that the effective dose of the antibody used for treatment may be increased or decreased during the particular course of treatment. Dosage changes result from the results of the diagnostic measures described herein and become apparent.

Thus, in one embodiment of the invention, the dosing regimen comprises the initial administration of a therapeutically effective dose of at least one anti-CD40 antibody in the treatment period of 1, 8, 15, and 22 days.

In another embodiment of the invention, the dosing regimen comprises a dosing regimen wherein the initial administration of a therapeutically effective dose of at least one anti-CD40 antibody is carried out daily or weekly, on days 1, 3, 5, and 7; Dosing regimen comprising the initial administration of a therapeutically effective dose of at least one anti-CD40 antibody at 1 and 3-4 days per week within the treatment period; And preferably a dosing regimen comprising first administering a therapeutically effective dose of at least one anti-CD40 antibody per day per week within the treatment period. The treatment period may include a period of one week, two weeks, three weeks, one month, two months, three months, six months, or one year. The treatment period can be continuous or separated, respectively, for 1 week, 2 weeks, 1 month, 3 months, 6 months, or 1 year.

In another embodiment of the invention, the initial therapeutically effective dose of an anti-CD40 antibody is defined as a subsequent dose corresponding to a high dosage range (ie, from about 20 mg / kg to about 50 mg / kg), as defined herein. Low dose range (ie, about 0.3 mg / kg to about 20 mg / kg).

In another embodiment of the invention, the initial therapeutically effective dose of an anti-CD40 antibody is defined as a subsequent dose corresponding to the low dosage range (ie, from about 0.3 mg / kg to about 20 mg / kg), as defined herein. High dose range (ie, about 20 mg / kg to about 50 mg / kg). Thus, in one embodiment of the invention, anti-CD40 antibody therapy is initiated by administering a “load dose” of the antibody to a subject in need thereof. "Load dose" means the initial dose of an anti-CD40 antibody administered to a subject, wherein the antibody dose administered is within the high dosage range (ie, from about 20 mg / kg to about 50 mg / kg). A “load dose” can be administered as a single dose, eg, a single infusion when the antibody is administered intravenously, or when multiple doses, eg, a complete “load dose” is administered within a period of about 24-hours. As long as the antibody is administered intravenously it is a multiple infusion. After administration of the “load dose”, the subject is administered a therapeutically effective dose of one or more additional anti-CD40 antibodies. Subsequent therapeutically effective dosages can be administered, for example, according to a weekly dosing schedule or once every two weeks, once every three weeks, or once every four weeks. In this embodiment of the invention, the subsequent therapeutically effective dose is generally in the low dose range (ie 0.3 mg / kg to about 20 mg / kg).

In another embodiment, in one embodiment of the invention, a subsequent therapeutically effective dose of an anti-CD40 antibody, in accordance with a "loading dose," may be administered according to a "maintenance plan," and the therapeutically effective dose of the antibody may be Once every month, once every 6 weeks, once every 2 months, once every 10 weeks, once every 3 months, once every 14 weeks, once every 4 months, every 18 Once every week, once every 5 months, once every 22 weeks, once every 6 months, once every 7 months, once every 8 months, once every 9 months, every 10 months Once every 1 month, once every 11 months, or once every 12 months. In this embodiment of the invention, the therapeutically effective dose of the anti-CD40 antibody is within the low dose range (ie 0.003 mg / kg to about 20 mg / kg), in particular subsequent doses at more frequent intervals, eg For example, once every two weeks to once every month, or in the high dosage range (ie, from about 20 mg / kg to about 50 mg / kg), especially subsequent doses at less frequent intervals For example, subsequent dosages may be administered separately, from about 1 month to about 12 months.

Anti-CD40 antibodies present in the pharmaceutical compositions described herein for use in the methods of the invention may be obtained by natural or recombinant techniques and may be used in mammalian sources such as mice, rats, rabbits, primates, pigs, and humans. It can be from any source, including. Preferably such polypeptides are derived from human sources, more preferably recombinant, human proteins of hybridoma cell lines.

Pharmaceutical compositions useful in the methods of the invention may include biologically active variants of the antagonist anti-CD40 antibodies of the invention as described herein.

Any pharmaceutical composition comprising an anti-CD40 antibody having the aforementioned binding properties as a therapeutically active ingredient can be used in the methods of the invention. Thus, liquid, lyophilized, or spray-dried compositions comprising one or more anti-CD40 antibodies can be prepared in an aqueous or non-aqueous solution or suspension for subsequent administration to a subject in accordance with the methods of the present invention. Each such composition will comprise at least one anti-CD40 antibody as a therapeutic or prophylactic active ingredient. A “therapeutically or prophylactically active ingredient” means that an anti-CD40 antibody is used to generate a desired therapeutic or prophylactic response to the treatment, prevention, or diagnosis of a disease or condition in a subject when the pharmaceutical composition is administered to the subject. It means specifically incorporated in the composition. Preferably, the pharmaceutical composition may comprise suitable stabilizers, fillers, or both to minimize problems with loss of protein stability and biological activity during manufacture and storage.

Formulations may be added with a pharmaceutical composition comprising an anti-CD40 antibody of the invention. Such formulations may include, but are not limited to, oils, polymers, vitamins, carbohydrates, amino acids, salts, buffers, albumin, surfactants, or fillers. Preferably carbohydrates include sugars or sugar alcohols such as monosaccharides, polysaccharides, or polysaccharides, or water soluble glucans. Sugars or glucans are fructose, glucose, mannose, sorbose, xylose, maltose, sucrose, dextran, flulan, dextrin, α and β cyclodextrins, soluble starch, hydroxyethyl starch, and carboxymethylcellulose Or mixtures thereof. "Sugar alcohol" is defined as C ₄ to C ₈ hydrocarbons including hydroxyl groups and includes galactitol, inositol, mannitol, xylitol, sorbitol, glycerol, and arabitol. These sugars or sugar alcohols may be used individually or in combination. The sugar or sugar alcohol concentration is 1.0% to 7% w / v., More preferably 2.0% to 6.0% w / v. Preferably the amino acids include carnitine, arginine, and betaine in the thyroid (L) form; However, other amino acids may be added. Preferred polymers include polyvinylpyrrolidone (PVP) having an average molecular weight of 2,000 to 3,000, or polyethylene glycol (PEG) having an average molecular weight of 3,000 to 5,000. Surfactants that can be added to the formulation are shown in EP 270,799 and 268,110.

In addition, antibodies can be chemically modified to increase their circulating half-life, eg, by covalent conjugation to a polymer. Preferred polymers, and methods of binding them to peptides, are described in US Pat. No. 4,766,106; 4,179,337; 4,495,285; And 4,609,546; They are integral and are incorporated herein by reference. Preferred polymers are polyoxyethylated polyols and polyethylene glycols (PEG). PEG is water soluble at room temperature and has the general formula: R (O--CH ₂ --CH ₂ ) n O--R, where R may be hydrogen or a protecting group such as an alkyl or alkanol group. Preferably, the protecting group has 1 to 8 carbons, more preferably it is methyl. The abbreviation n is a positive integer, preferably 1 to 1,000, more preferably 2 to 500. PEG has an average molecular weight of preferably 1,000 to 40,000, more preferably 2,000 to 20,000, most preferably 3,000 to 12,000. Preferably, PEG has at least one hydroxy group, more preferably a terminal hydroxy group. This hydroxy group is preferably activated to react with the free amino group on the inhibitor. However, it should be understood that the type and amount of reactor may vary to achieve covalently conjugated PEG / antibodies of the invention.

Water soluble polyoxyethylated polyols are also useful in the present invention. These include polyoxyethylated sorbitol, polyoxyethylated glucose, polyoxyethylated glycerol (POG), and the like. POG is preferred. This is because the glycerol backbone of polyoxyethylated glycerol is the same backbone naturally occurring in mono-, di-, triglycerides, for example in animals and humans. Thus, such branching need not be seen as an outpatient agent in the body. POG preferably has a molecular weight in the same range as PEG. The structure of POG is described in Knauf et al. (1988) J Bio. Chem. 263: 15064-15070, a discussion of POG / IL-2 conjugation can be found in US Pat. No. 4,766,106, all of which are incorporated herein by reference in their entirety.

Another drug delivery system that increases circulating half-life is liposomes. Methods for preparing liposome delivery systems are described in Gabizon et al. (1982) Cancer Research 42: 4734; Cafiso (1981) Biochem Biophys Acta 649: 129; And Szoka (1980) Ann. Rev. Biophys. Eng. Introduced at 9: 467. Other drug delivery systems are also known and are described, for example, in Poznansky et al. (1980) Drug Delivery Systems (R. L. Miano, ed., Oxford, N. Y.) pp. 253-315; Poznansky (1984) Pharm Revs 36: 277.

Formulations incorporated into pharmaceutical compositions should provide stability of the anti-CD40 antibody. That is, the anti-CD40 antibodies must retain their physical and / or chemical stability and the desired biological activity, ie one or more antagonist activity as defined herein, wherein normal human peripheral B cells stimulated by T cells. Inhibition of immunoglobulin secretion by; Inhibition of survival and / or proliferation of normal human peripheral B cells stimulated by Jurkat T cells; Inhibition of survival and / or proliferation of normal human peripheral B cells stimulated by CD40L-expressing cells or soluble CD40 ligands (sCD40L); inhibition of "survival" anti-apoptotic intracellular signals in all cells stimulated by sCD40L or solid-phase CD40L; inhibition of CD40 signal transduction in all cells ligated with sCD40L or solid-phase CD40L; And inhibition of proliferation of human malignant B cells as referred to herein.

Methods of monitoring protein stability are well known in the art. For example, Jones (1993) Adv. Drug Delivery Rev. 10: 29-90; Lee, ed. (1991) Peptide and Protein Drug Delivery (Marcel Dekker, Inc., New York, New York); See the stability measurement method disclosed herein. In general, protein stability is measured for a specific period of time at a selected temperature. In a preferred embodiment of the invention, the stable antibody pharmaceutical formulation provides stability of the anti-CD40 antibody when stored at room temperature (about 25 ° C.) for at least 1 month, at least 3 months, or at least 6 months, and / or It is stable upon storage at about 2-8 ° C. for at least 6 months, at least 9 months, at least 12 months, at least 18 months, and at least 24 months.

When a protein such as an antibody is formulated into a pharmaceutical composition, a visual signal of precipitation, aggregation, and / or denaturation in the pharmaceutical composition (ie, discoloration or loss of clarity) or a measurable signal (eg, size- If exclusion chromatography (SEC) or UV light scattering is used, it was considered to retain their physical stability at a given time point. With regard to chemical stability, when a protein such as an antibody is formulated into a pharmaceutical composition, the measurement of chemical stability indicates that the protein (ie, antibody) retains the biological activity of interest in the pharmaceutical composition, given a time point. It was considered to retain their chemical stability at. Methods of monitoring changes in chemical stability are well known in the art and include, for example, clipping using SDS-PAGE, SEC, and / or matrix-assisted laser desorption ionization / flight time mass spectrometry; Examples include, but are not limited to, methods of detecting chemically modified forms of proteins as a result of degradation associated with changes in molecular charge (eg, deaminoation) using ion-exchange chromatography. For example, this method is disclosed herein.

When pharmaceutical compositions are formulated, it is contemplated that the anti-CD40 antibody retains the desired biological activity at a given time point. If the desired biological activity at that time is within about 30%, preferably within about 20% of the desired biological activity within that time, then the pharmaceutical composition is determined in a suitable assay for the desired biological activity. Are manufactured. Assays to determine the desired biological activity of an anti-CD40 antibody can be performed as described in the Examples herein. Schultze et al. (1998) Proc. Natl. Acad. ScL USA 92: 8200-8204; Denton et al. (1998) Pediatr. Transplant. 2: 6-15; Evans et al. (200O) J Immunol. 164: 688-697; Noelle (1998) Agents Actions Suppl. 49: 17-22; Lederman et al. (1996) Curr. Opin. Hematol. 3: 77-86; Coligan et al. (1991) Current Protocols in Immunology 13:12; Kwekkeboom et al. (1993) Immunology 79: 439-444; And the measurements described in US Pat. Nos. 5,674,492 and 5,847,082; It is incorporated herein by reference.

In one embodiment of the invention, the anti-CD40 antibody is formulated in a liquid pharmaceutical formulation. Anti-CD40 antibodies can be prepared using any known method and include the methods disclosed herein. In one embodiment of the invention, anti-CD40 antibodies were produced recombinantly in CHO cell lines.

If the anti-CD40 antibodies are stored prior to their formulation, they can be frozen, for example thawing at room temperature for ≦ -20 ° C. and then for further formulation. Liquid pharmaceutical formulations comprise a therapeutically effective amount of an anti-CD40 antibody. The amount of these antibodies present in the formulation takes into account the route of administration and the desired volume of administration.

In this manner, the liquid pharmaceutical composition may contain about 0.1 mg / ml to about 50.0 mg / ml, about 0.5 mg / ml to about 40.0 mg / ml, about 1.0 mg / ml to about 30.0 mg / ml anti-CD40 antibody. , From about 5.0 mg / ml to about 25.0 mg / ml, from about 5.0 mg / ml to about 20.0 mg / ml, or from about 15.0 mg / ml to about 25.0 mg / ml. In one embodiment of the present invention, the liquid pharmaceutical composition comprises about 0.1 mg / ml to about 5.0 mg / ml, about 5.0 mg / ml to about 10.0 mg / ml, about 10.0 mg / ml to about 15.0 mg / ml, about 15.0 mg / ml to about 20.0 mg / ml, about 20.0 mg / ml to about 25.0 mg / ml, about 25.0 mg / ml to about 30.0 mg / ml, about 30.0 mg / ml to about 35.0 mg / ml, about 35.0 mg anti-CD40 antibody at a concentration of / ml to about 40.0 mg / ml, about 40.0 mg / ml to about 45.0 mg / ml, or about 45.0 mg / ml to about 50.0 mg / ml. In another embodiment of the invention, the liquid pharmaceutical composition comprises about 15.0 mg / ml, about 16.0 mg / ml, about 17.0 mg / ml, about 18.0 mg / ml, about 19.0 mg / ml, about 20.0 mg / ml, about Anti-CD40 antibody at a concentration of 21.0 mg / ml, about 22.0 mg / ml, about 23.0 mg / ml, about 24.0 mg / ml, or about 25.0 mg / ml. Liquid pharmaceutical compositions include anti-CD40 antibodies and about pH 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, other such values include buffers to maintain the pH of the formulation in the range of about pH 5.0 to about pH 7.0 in the range of about pH 5.0 to about pH 7.0. In one embodiment of the invention, the buffer has a pH of the formulation of about pH 5.0 to about pH 6.5, about pH 5.0 to about pH 6.0, about pH 5.0 to about pH 5.5, about pH 5.5 to about 7.0, about pH 5.5 to about pH 6.5, or about pH 5.5 to about pH 6.0.

As described above, any suitable buffer that maintains the pH of the liquid anti-CD40 antibody formulation in the range of about pH 5.0 to about pH 7.0, to the extent that it retains the physicochemical stability and the desired biological activity of the antibody, can be used in the formulation. Suitable buffers include, but are not limited to, conventional acids and salts thereof, wherein the counterion can be, for example, sodium, potassium, ammonium, calcium, or magnesium. Examples of conventional acids and salts thereof that can be used as buffers in pharmaceutical liquid formulations are succinic or succinate, citric acid or citrate, acetic acid or acetate, tartaric acid or tartarate, phosphoric acid or phosphate, gluconic acid or gluconate , Glutamic acid or glutamate, aspartic acid or aspartate, maleic acid or maleate, and malic acid or malate buffers. The buffer concentration in the formulation can be about 1 mM to about 50 mM, and about 1 mM, 2 mM, 5 mM, 8 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM , 45 mM, 50 mM, or other value within the range of about 1 mM to about 50 mM. In one embodiment of the invention, the buffer concentration in the formulation is about 5 mM to about 15 mM, and about 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, or other value within the range of about 5 mM to about 15 mM.

In one embodiment of the invention, the liquid pharmaceutical formulation comprises a succinic acid at a concentration that maintains a therapeutically effective amount of the anti-CD40 antibody and the pH of the formulation at about pH 5.0 to about pH 7.0, preferably about pH 5.0 to about pH 6.5. Salt buffer or citrate buffer. "Succinate buffer" or "citrate buffer" means a buffer comprising each of a salt of succinic acid or a salt of citric acid. In a preferred embodiment of the present invention, the succinate or citrate counterions are sodium cations and thus the buffers are sodium succinate or sodium citrate, respectively. However, all cations are expected to be effective. Other possible succinate or citrate cations include but are not limited to potassium, ammonium, calcium, and magnesium. As mentioned above, the succinate or citrate buffer concentration in the formulation can be about 1 mM to about 50 mM, and about 1 mM, 2 mM, 5 mM, 8 mM, 10 mM, 15 mM, 20 mM, 25 mM , 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, or other values ranging from about 1 mM to about 50 mM. In one embodiment of the invention, the buffer concentration in the formulation is about 5 mM to about 15 mM, and about 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, or about 15 mM. In another embodiment of the invention, the liquid pharmaceutical formulation comprises an anti-CD40 antibody at a concentration of about 0.1 mg / ml to about 50.0 mg / ml, or about 5.0 mg / ml to about 25.0 mg / ml, and about 1 mM to Succinate or citrate buffers, such as sodium succinate or sodium citrate buffer, at a concentration of about 20 mM, about 5 mM to about 15 mM, preferably about 10 mM.

If a liquid pharmaceutical formulation that is in close proximity isotonic is desired, the liquid pharmaceutical formulation comprising an anti-CD40 antibody and a buffer may further comprise an amount of isotonic agent sufficient to impart close isotonicity to the formulation. "Proximity isotonic" means that the aqueous formulation has about 240 mmol / kg to about 360 mmol / kg, preferably about 240 to about 340 mmol / kg, more preferably about 250 to about 330 mmol / kg, even more preferably Is meant to have an osmolarity of about 260 to about 320 mmol / kg, more preferably about 270 to about 310 mmol / kg. Methods of determining the isotonicity of a solution are known to those skilled in the art. See, eg, Setnikar et al. (1959) J Am. Pharm. Assoc. See 48: 628.

Those skilled in the art are familiar with the various pharmaceutically acceptable solutes useful for providing isotonicity in pharmaceutical compositions. Isotonic agents can be any reagent that can control the osmotic pressure of the liquid pharmaceutical formulation of the invention to a level equivalent to body fluids. Preference is given to using physiologically acceptable isotonic agents. Thus, liquid pharmaceutical formulations comprising a therapeutically effective amount of an anti-CD40 antibody and a buffer may include components that can be used to provide isotonicity, such as sodium chloride; Amino acids such as alanine, valine, and glycine; Sugars and sugar alcohols (polyols), including but not limited to glucose, dextrose, fructose, sucrose, maltose, mannitol, trehalose, glycerol, sorbitol, and xylitol; Acetic acid, other organic acids or salts thereof, and relatively small amounts of citrate or phosphate salts. One skilled in the art recognizes additional agents suitable for providing the stiffness of liquid formulations.

In one preferred embodiment of the invention, the liquid pharmaceutical formulation comprising an anti-CD40 antibody and a buffer may further comprise sodium chloride as an isotonic agent. The sodium chloride concentration in the formulation will depend on the contribution of other components to the stiffness. In one embodiment of the invention, the concentration of sodium chloride is from about 50 niM to about 300 mM, about 50 mM to about 250 mM, about 50 mM to about 200 mM, about 50 mM to about 175 mM, about 50 mM to about 150 mM, about 75 mM to about 175 mM, about 75 mM to about 150 mM, about 100 mM to about 175 mM, about 100 mM to about 200 mM, about 100 mM to about 150 mM, about 125 mM to about 175 mM , About 125 mM to about 150 mM, about 130 mM to about 170 mM, about 130 mM to about 160 mM, about 135 mM to about 155 mM, about 140 mM to about 155 mM, or about 145 mM to about 155 mM . In one embodiment of the invention, the concentration of sodium chloride is about 150 mM. In another embodiment of the invention, the concentration of sodium chloride is about 150 mM, the buffer is sodium succinate or sodium citrate buffer at a concentration of about 5 mM to about 15 mM, and the liquid pharmaceutical formulation is an anti-CD40 antibody. A therapeutically effective amount of and a pH of about pH 5.0 to about pH 7.0, about pH 5.0 to about pH 6.0, or about pH 5.5 to about pH 6.5. In another embodiment of the invention, the liquid pharmaceutical formulation comprises an anti-CD40 antibody at a concentration of about 0.1 mg / ml to about 50.0 mg / ml or about 5.0 mg / ml to about 25.0 mg / ml, about 150 mM sodium chloride. And about 10 mM sodium succinate or sodium citrate at a pH of about pH 5.5.

Proteolysis based on freeze thawing or mechanical shearing in the course of the liquid pharmaceutical formulation of the present invention can be inhibited by incorporating a surfactant into the formulation to lower the surface tension at the solution-air interface. Thus, in one embodiment of the invention, the liquid pharmaceutical formulation comprises a therapeutically effective amount of an anti-CD40 antibody, a buffer, and additionally comprises a surfactant. In another embodiment of the invention, the liquid pharmaceutical formulation comprises an anti-CD40 antibody, a buffer, an isotonic agent, and further comprises a surfactant.

Typical surfactants used are nonionic surfactants, including polyoxyethylene sorbitol esters such as polysorbate 80 (Tween 80) and polysorbate 20 (Tween 20); Polyoxypropylene-polyoxyethylene esters such as Pluronic F68; Polyoxyethylene alcohols such as Brij 35; Simethicone; Polyethylene glycols such as PEG400; Lysophosphatidylcholine; And polyoxyethylene-p-t-octylphenols such as Triton X-100. Stabilization of traditional agents by surfactants or emulsifiers is described, for example, in Levine et al. (1991) J Parenteral Sci. Technol. 45 (3): 160-165, which is incorporated herein by reference. A preferred surfactant used in the practice of the present invention is polysorbate 80. When surfactant is included, it is generally from about 0.001% to about 1.0% (w / v), from about 0.001% to about 0.5%, from about 0.001% to about 0.4%, from about 0.001% to about 0.3%, from about 0.001% to About 0.2%, about 0.005% to about 0.5%, about 0.005% to about 0.2%, about 0.01% to about 0.5%, about 0.01% to about 0.2%, about 0.03% to about 0.5%, about 0.03% to about 0.3 %, About 0.05% to about 0.5%, or about 0.05% to about 0.2%.

Thus, in one embodiment of the present invention, the liquid pharmaceutical formulation comprises a therapeutically effective amount of an anti-CD40 antibody and the buffer is about 1 mM to about 50 mM, about 5 mM to about 25 mM, or about 5 mM to Sodium succinate or sodium citrate buffer at a concentration of about 15 mM; The formulation has about pH 5.0 to about pH 7.0, about pH 5.0 to about pH 6.0, or about pH 5.5 to about pH 6.5; The formulation comprises a surfactant such as polysorbate 80 in an amount of about 0.001% to about 1.0% or about 0.001% to about 0.5%. Such formulations may optionally include isotonic agents, such as sodium chloride, at a concentration of about 50 mM to about 300 mM, about 50 mM to about 200 mM, or about 50 mM to about 150 mM. In another embodiment of the invention, the liquid pharmaceutical formulation comprises an anti-CD40 antibody at a concentration of about 0.1 mg / ml to about 50.0 mg / ml or about 5.0 mg / ml to about 25.0 mg / ml, about 20.0 includes mg / ml; Comprises sodium chloride at a concentration of about 50 mM to about 200 mM and comprises about 150 mM sodium chloride; Sodium succinate or sodium citrate at a concentration of about 5 mM to about 20 mM, and about 10 mM sodium succinate or sodium citrate; The concentration of sodium chloride is from about 50 mM to about 200 mM and comprises about 150 mM; Optionally a surfactant such as polysorbate 80, comprising from about 0.001% to about 1.0% and from about 0.001% to about 0.5%; In this case, the liquid pharmaceutical formulation may have a pH of about pH 5.0 to about pH 7.0, about pH 5.0 to about pH 6.0, about pH 5.0 to about pH 5.5, about pH 5.5 to about pH 6.5, or about pH 5.5 to about pH 6.0. Have

Liquid pharmaceutical formulations may essentially lack any of the preservatives and other carriers, excipients, or stabilizers described above. In other embodiments, the formulation includes one or more preservatives, such as antibacterial agents, pharmaceutically acceptable carriers, excipients, or stabilizers described above that do not adversely affect the stability of physicochemical anti-CD40 antibodies. can do. Examples of acceptable carriers, excipients, and stabilizers include, but are not limited to, additional buffers, cosolvents, surfactants, antioxidants including ascorbic acid and methionine, chelating agents such as EDTA, metal complexes (eg, Zn-protein complexes) , And biodegradable polymers such as polyesters. After a thorough discussion of formulations, the selection of pharmaceutically acceptable carriers, stabilizers, and isomolytes can be found in Remington's Pharmaceutical Sciences (18th ed .; Mack Publishing Company, Eaton, Pennsylvania, 1990). Which is incorporated herein by reference.

As used herein, “carrier” includes pharmaceutically acceptable carriers, excipients, or stabilizers that are nontoxic to cells or mammals that are exposed to them at the dosages and concentrations employed. Physiologically acceptable carriers are generally aqueous pH buffers. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, succinate, and other organic acids; Antioxidants including ascorbic acid; Low molecular weight (up to about 10 residues) polypeptides; Proteins such as serum albumin, gelatin, or immunoglobulins; Hydrophilic polymers such as polyvinylpyrrolidone; Amino acids such as glycine, glutamine, asparagine, arginine or lysine; Monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; Chelating agents such as EDTA; Sugar alcohols such as mannitol or sorbitol; Salt-forming counterions such as sodium; And / or nonionic surfactants such as TWEEN, polyethylene glycol (PEG), and pluronic.

Administration “in combination” with one or more additional therapeutic agents includes simultaneous and continuous administration in any order.

After preparation of the liquid pharmaceutical formulations or other pharmaceutical compositions described herein, they may be lyophilized to prevent degradation. Lyophilization methods of liquid compositions are known to those skilled in the art. Immediately before use, the composition may be reconstituted with sterile diluent (ringer solution, distilled water, or sterile saline, for example) containing additional components. Upon reconstitution, the composition is preferably administered to a subject using methods known to those skilled in the art.

In one embodiment of the invention, the anti-CD40 antibody comprises at least one antibody, including but not limited to surgery, radiation therapy, chemotherapy, cytokine therapy, or other monoclonal antibodies intended for use in treating a subject solid tumor It can be administered in combination with other cancer therapies, and additional cancer therapies can be administered before, during or after the anti-CD40 antibody therapy. Thus, when the administration of an anti-CD40 antibody to combined administration is combined with the administration of another therapeutic agent, or another monoclonal antibody as chemotherapy, cytokine therapy, the method of the present invention is coadministered using a separate formulation or a single pharmaceutical formulation. , And sequential sequential administration, where it is desirable to have time for two (or all) active agents to exert their therapeutic activity simultaneously. If the methods of the invention include a combined treatment regimen, such therapies may be given simultaneously. That is, the anti-CD40 antibody is administered as another cancer therapy at the same time or within the same time period (ie, the therapy is performed simultaneously, but the anti-CD40 antibody is not administered as another cancer therapy at the same time). In other embodiments, anti-CD40 antibodies of the invention may also be administered prior to or subsequent to other cancer therapies. Sequential administration of other cancer therapies may be performed regardless of whether the treated patient responds to the first course of therapy to reduce the likelihood of palliation or relapse. If the combination therapy comprises administering an anti-CD40 antibody in combination with administration of a cytotoxin, it is preferred that the anti-CD40 is administered prior to the administration of the cytotoxin.

In this manner, the anti-CD40 antibody is administered in combination with at least one other cancer therapy, including surgical or surgical methods (eg, splenectomy, liver resection, lymphadenectomy, leukocyte transfer, bone marrow transplant, etc.); radiotherapeutics; Chemotherapy, including but not limited to combinations with autologous bone marrow transplantation, wherein suitable chemotherapeutic agents are fludarabine or fludarabine phosphate, chlorambutyl, vinkrastin, pentostatin, 2-chlorodeoxyadenosine (Cladribine), cyclophosphamide, doxorubicin, prednisone, and combinations thereof, such as anthracycline-containing therapies such as CAP (cyclophosphamide, doxorubicin plus prednisone), CHOP (cyclophosphamide, Vinkrastin, prednisone plus doxorubicin), VAD (vincristine, doxorubicin, plus dexamethasone), MP (melphalan plus prednisone), and other cytotoxic and / or therapeutic agents used in chemotherapy such as mitoxantrone, daunoru Bicine, idarubicin, asparaginase, and cytarabine, methotrexate, 5-fluorouracil decarbazine, 6-thioguanine, 6-tomer Anti-metabolites, purine, and including, but not limited to Nella Rabin; Other anti-cancer monoclonal antibody therapies (eg, Alemtuzumab (Campath®) or other anti-CD52 antibody targeting CD52 cell-surface glycoproteins on malignant B cells; Rituxan®, a whole human antibody HuMax- CD20, R-1594, IMMU-106, TRU-015, AME-133, Tocitumomab / I-131 Tocitumomab (Bexxar®), Ibritumomab Thiuxetane (Zevalin®), or CD20 on malignant B cells All other therapeutic anti-CD20 antibodies targeting antigens; anti-CD19 antibodies (eg, MT103, bispecific antibodies); anti-CD22 antibodies (eg, humanized monoclonal antibody epratuzumab); bevacizu Mab (Avastin®) or other anticancer antibody targeting human vascular endothelial growth factor; anti-CD22 antibody targeting CD22 antigen on malignant B cells (eg monoclonal antibody BL-22, alphaCD22 toxin); macrophage colon Α-M-CSF antibodies targeting stimulating factors; nuclear factor-kappaB overexpressed in multiple myeloma Antibodies that target receptor activators of (RANK) and their ligands (RANKL); anti-CD23 antibodies that target CD23 antigens on malignant B cells (eg, IDEC-152); anti-CD80 that targets CD80 antigens Antibodies (eg, IDEC-114); anti-CD38 antibodies targeting CD38 antigen on malignant B cells; antibodies targeting major histocompatibility complex class II receptors expressing malignant B cells (anti-MHC antibodies); malignant Other anti-CD40 antibodies targeting CD40 antigen on B cells (eg SGN-40); and antibodies targeting tumor necrosis factor-associated apoptosis-inducing ligand receptor 1 (TRAIL-Rl) (eg , Agonistic human monoclonal antibody HGS-ETRl) and TRAIL-R2 expressed in a number of solid tumors and tumors of hematological origin; small molecule-based cancer therapies such as microtubules and / or topoisomerase inhibitors (eg Mitosis inhibitor dorastatin And dorastatin analogs; Tubulin-binding agent T900607; XLl 19; And topoisomerase inhibitor aminocamptothecin), SDX-105 (bendamustine hydrochloride), isabepilone (epothilone analogue, referred to as BMS-247550), protein kinase C inhibitors such as midostaurine ( (PKC-412, CGP 41251, N-benzoylsutaurosporin), pisanthrone, eloxatin (antitumor), ganite (gallium nitric acid), Thalomid® (thalidomide), immunoregulatory derivatives of thalidomide , Levimid (formerly levimid)), Affinitak ™ (antisense inhibitor of protein kinase C-alpha), SDX-101 (R-etodolak, induces apoptosis of malignant lymphocytes), second generation purine nucleosides Analogs such as cloparabine, inhibitors of the production of protein Bcl-2 by cancer cells (eg antisense oblimersen and Genasense®), proteasome inhibitors (eg Velcade ™ (bortezomib)), Small molecule kinase inhibitors (eg , CHIR-258), small molecule VEGF inhibitors (eg ZD-6474), small molecule inhibitors of heat shock protein (HSP) 90 (eg 17-AAG), histone deacetylases (eg , Hybrid / polar cell differentiation HPC) preparations such as suberanilohydroxamic acid (SAHA), and small molecule inhibitors and apoptosis agents of FR-901228 such as Trisenox® (arsen trioxide) and Xcytrin® (methoxapine) Small molecule-based cancer therapy, including but not limited to, gadolinium); vaccine approaches (e.g., Id-KLH, oncophage, vitaletatin), personalized immunotherapy, or active neotypical immunotherapy (e.g., MyVax® personalized immunotherapy, previously designated GTOP-99), Promune® (CpG 7909, a synthetic agent of Toll-like receptor 9 (TLR9)), interferon-alpha therapy, interleukin-2 (IL-2) therapy, IL Vaccine / immunotherapy-based cancer therapy, including but not limited to, -12 therapy, IL-15 therapy, and IL-21 therapy; De therapy; Or other cancer therapies; Wherein the additional cancer therapy is administered prior to, along with or following the antagonist anti-CD40 antibody therapy.

In one embodiment of the invention, the anti-CD40 antibody is administered in combination with Bortezomib (VELCADE®), particularly for the treatment of multiple myeloma. WO 2005/044855 A2 discloses that the combined treatment of CHIR-12.12 with Bortezomib increases tumor growth inhibition efficacy in multiple myeloma experimental models.

In one embodiment of the invention, the anti-CD40 antibody is administered in combination with IL-2, in particular for the treatment of B cell lymphoma. WO 2005/044294 A2 discloses that the combined treatment of CHIR-12.12 with IL-2 resulted in additional anti-tumor activity against Namalwa tumors.

Accordingly, the present invention provides anti-CD40 in the manufacture of a medicament for the treatment of cancer or precancerous conditions associated with CD40-expressing cells of human patients that are heterozygous or homozygous (genotype V / F or F / F) to FcγRJIIa-158F. A therapeutic or prophylactically effective amount of an antibody is provided, wherein the medicament is in harmony with the treatment of at least one other cancer therapy.

By “harmonized” it is meant that a medicament comprising an anti-CD40 antibody can be used before, during or after treatment of a subject with at least one other cancer therapy.

The invention also provides the use of an anti-CD40 antibody in the manufacture of a medicament for treating a human patient in a cancer or precancerous condition related to CD40-expressing cells, wherein the human patient is heterozygous or homozygous for FcγRIIIa-158F. Genotype V / F or F / F) and were pretreated with at least one other tumor treatment.

"Pretreated" or "pretreatment" means that one or more other cancer therapies have been received (ie, have been treated with at least one other cancer therapy) prior to administration of a medicament comprising an anti-CD40 antibody. “Pretreated” or “pretreatment” means within 2 years, within 18 months, within 1 year, within 6 months, within 2 months, with at least one other cancer therapy prior to the initiation of the treatment of a medicament comprising an anti-CD40 antibody, Treated within 6 weeks, within 1 month, within 4 weeks, within 3 weeks, within 2 weeks, within 1 week, within 6 days, within 5 days, within 4 days, within 3 days, 2 days, or even within 1 day It includes a subject. The subject need not be a prior cancer therapy, or a responder of pretreatment with prior cancer therapies. Thus, a subject receiving a medicament comprising an antagonist anti-CD40 antibody may or may not have responded (ie, has not responded to) prior cancer therapy, or one or more prior cancer therapies consisting of multiple cancer therapies. Is resistant). Examples of other cancer therapies in which a subject may be pretreated before administration of a medicament comprising an anti-CD40 antibody include surgery; radiotherapeutics; Chemotherapy, including but not limited to combinations with autologous bone marrow transplantation, wherein suitable chemotherapeutic agents include those described herein and WO 2005/044854, WO 2005/044304, WO 2005/044305, WO 2005/044306, Including but not limited to those described in WO 2005/044855, WO 2005/044307, and WO 2005/044294; Other anticancer monoclonal antibody therapies are described in the anticancer antibodies described herein and in WO 2005/044854, WO 2005/044304, WO 2005/044305, WO 2005/044306, WO 2005/044855, WO 2005/044307, and WO 2005/044294. Includes, but is not limited to; Small molecule-based cancer therapy is described in the small molecules described herein and in WO 2005/044854, WO 2005/044304, WO 2005/044305, WO 2005/044306, WO 2005/044855, WO 2005/044307, and WO 2005/044294. Including but not limited to those described; Vaccine / immunotherapy-based cancer therapy includes those described herein and WO 2005/044854, WO 2005/044304, WO 2005/044305, WO 2005/044306, WO 2005/044855, WO 2005/044307, and WO 2005/044294. Including but not limited to those described in; Steroid therapy; Other cancer therapies; Or any combination thereof.

"Treatment" as a harmonization of an anti-CD40 antibody with one or more other cancer therapies is herein used to treat, cure, alleviate, alleviate, alter, cure, ameliorate, ameliorate a disease, symptom of a disease, or propensity for a disease. Or the application or administration of an anti-CD40 antibody or other cancer therapy to a patient having a disease, symptom of disease, or propensity for disease, or tissue or cell line isolated from the patient for the purpose of affecting it. "Treatment" refers to a disease, symptom, or disposition for a disease, for the purpose of treating, healing, alleviating, alleviating, altering, treating, improving, improving, or affecting a disease, symptom, or disposition for the disease. Applying or administering a pharmaceutical composition or other cancer therapy comprising an anti-CD40 antibody to a patient, or applying a pharmaceutical composition or other cancer therapy comprising an anti-CD40 antibody to a tissue or cell line isolated from the patient Or administration.

In one embodiment of the present invention, a combination therapy provides a synergistic improvement in therapeutic efficacy over an individual treatment administered alone. The term "synergy" is used to describe the combined effect of two or more active agents that is greater than the sum of the individual effects of each individual active agent. Thus, when the binding effect of two or more agents is indicative of activity or progression, eg, "synergistic" inhibition of tumor growth, it means that the inhibition of activity or progression is greater than the sum of the inhibitory effects of each individual active agent. . The term “synergistic therapeutic effect” refers to a therapeutic effect observed in a combination of two or more therapies, where the therapeutic effect (as measured by a number of variables) is the sum of the individual therapeutic effects observed by the individual therapies. Greater than

Various features and implementations of the invention will be described in more detail by way of example. It is apparent that such specific changes can be made without departing from the scope of the present invention.

Experiment

The anti-CD40 antibody used in the following experiment is CHIR-12.12. The production, sequence and properties of the CHIR-12.12 antibody can be found in published international patent applications WO 2005/044854, WO 2005/044304, WO 2005/044305, WO 2005/044306, WO 2005/044855, WO 2005/044307, and WO 2005/044294. It is explained in detail in. Hybridoma strain 153.8E2.D10.D6.12.12 (CMCC # 12056), which expresses a CHIR-12.12 antibody, has an American type culture selection [ATCC; 10801 University Blvd., Manassas, Virginia 20110-2209 (USA), deposited as patent accession number PTA-5543.

Example  1: within cell lines ADCC Analysis of

Including lymphoma cell lines (Daudi, Narnalwa), multiple myeloma cell lines (ARH77, IM-9), B-ALL cell line (CCRF-SB), and B-CLL cell line (EHEB) for CHIR-12.12 and rituximab Their relative ADCC activity against various malignant B-cell lines expressing both CD40 and CD20 antigens was compared.

The ADCC effects and titers measured as the maximum percentage lysis and ED50 for CHIR-12.12 and Rituximab, respectively, were compared. The experimental results are shown in FIGS. 1A-IF. For all target cell lines, CHIR-12.12 is a more potent, potent mediator of ADCC than rituximab. In the test of 6 cell lines, the number of cell surface CD20 molecules per cell was 2.6-30.8 times higher than CD40. These data indicate that malignant B-cell lines are more effectively lysed by CHIR-12.12 than rituximab, even though CD40 molecules are expressed less than CD20.

Example  2: CLL  Within patient cells ADCC Analysis of

Relative ADCC activity of CHIR-12.12 and rituximab on 8 in vitro primary CLL cells was compared. CHIR-12.12 showed greater ADCC than rituximab for all patients' CLL (FIGS. 2A-D and 3). Mean percentage maximal dissolution by CHIR-12.12 and Rituximab was 49 ± 16% and 31 ± 14%, respectively. CHIR-12.12 was 10 times more potent than rituximab as measured by ED50 values (14.1 pM vs 155.5 pM, respectively).

Antibody-Dependent Cell Cytotoxicity ( ADCC ) Experimental Design

Target cell: CLL patient cell, 5000 / well. Effector cells: purified normal human NK cells, 50,000 / well. E: T ratio: 10. Abs concentrations: 0.00001, 0.0001, 0.001, 0.01, 0.1, 1 and 10 μg / ml. Incubation time: 4 hours. Culture medium: RPMI (w / o Phenol red) + 10% FBS + 1% P / S. Incubator: 96-well round bottom plate. Read: Calcein AM emission measured by Arbitrary Fluorescence Unit (AFU) with 485 nm excitation / 535 nm emission. Calculation:% specific lysis = 100 x (AFU test-AFU spontaneous release 1) / (AFU maximal release 2-AFU spontaneity). Negative control: calcein released by target cells in the absence of antibody or NK cells. Positive Control: Calcein released by target cells lysed by detergent (1% NP40).

The results shown in FIGS. 2 and 3 show that CHIR-12.12 mediated ADCC more than rituximab for CLL patient cells. The magnitude of the ADCC difference depends on whether it is a target cell or an NK donor cell, but was observed for all patient samples. When CLL cells from a single patient were tested against two different NK donors, CHIR-12.12 mediated ADCC more than rituximab for both NK donor cells, although the magnitudes of the ADCC differences were not the same (FIG. 4). ). The mechanical basis of this superior ADCC may include the relative expression levels of target antigens (CD20 and CD40), the degree of antibody internalization, and antibody affinity for FcγIIIa receptors on NK cells. Thus the effect of these factors on the ADCC activity of CHIR-12.12 and Rituximab was investigated.

Example  3: cell-surface CD40  And CD20  Quantification of Molecules

CD20 and CD40 quantitative densities on CLL cells (Example 3) and degree of antibody internalization (Example 4) were investigated for potential causes of the differences in ADCC activity described above. Relative ADCC activities of CHIR-12.12 and rituximab on 9 in vitro primary CLL cells were compared. CHIR-12.12 showed more ADCC than rituximab for all patients' CLL (FIGS. 2A-D and 3). Mean percentage maximal dissolution by CHIR-12.12 and Rituximab was 48 ± 15% and 30 ± 14%, respectively. CHIR-12.12 was 10 times more potent than rituximab as measured by ED50 values (13.2 pM vs. 147.2 pM, respectively, FIG. 6). Since the number of CD20 is 1.3 to 14 times greater than the CD40 molecules on the cell surface, the greater ADCC activity and efficacy of CHIR-12.12 does not depend on the high density of the cell surface CD40 molecules (FIGS. 5 and 6).

Way

Cells were precultured in staining buffer (containing PBS, 1% BSA, 0.1% Na azide) with 1 mg / ml human IgG1 to block non-specific binding sites. These were incubated at 4 ° C. (ice) for 30 minutes. FITC-conjugated human IgG1 isotype control, FITC-conjugated CHIR-12.12, or FITC-conjugated rituximab were added at 100, 10, 1, 0.1 μg / ml and the cells were added for 30 minutes. Incubated at 4 ° C. (ice). Cells were washed with staining buffer (PBS + l% FBS + 0.1% sodium azide) and analyzed by FACS caliber.

Geometric mean fluorescence intensity was measured by FACS. Isosoluble fluorescent chromosome molecules (MESFs) are then calculated based on standard curves set by adjusted FITC beads.

Example  4: CH12 .12 on cell lines CD40 By binding to Internalization  Do not induce

Daudi, lymphoma cell line, and ARH77, MM cell line are used to assess the effect of CH 12.12 binding on internalization. Cells are incubated with human IgG1 (control antibody) or incubated for 3 hours with ice (0.1% sodium azide addition, no internalization) or 37 ° C. (no sodium azide) with 1 μg / ml CH12.12 do. After washing with cold staining buffer (PBS + 1% BSA + 0.1% sodium azide), cells are stained in ice for 30 minutes with goat anti-human IgG-FITC. Geometric mean fluorescence intensity (MFI) was recorded by FACS caliber. It was observed that there was no difference in MFI between cells incubated with CH 12.12 at 37 ° C. on ice in the presence of sodium azide or in the absence of sodium azide (FIG. 7). These data show that for binding of CD40, CH12.12 is not internalized and continues to manifest on the cell surface.

Example  5: CLL  After binding to patient cells CHIR -12.12 and Of rituximab  Internalization: FACS  And Confocal  microscope

Confocal  Microscopy methodology

Cells were treated with Alexa 488 or FITC conjugated CHIR-12.12, Rituximab, and IgG1 10 μg / ml for 3 hours at 40 ° C. (with 0.1% Na azide) or 37 ° C. (without Na azide). And incubated. The cells were then washed and fixed with 2% formaldehyde, 5 min RT. Cells were then washed, placed on poly-L-lysine coated slides, mounted, sealed and analyzed by confocal imaging.

result

The results of these experiments are shown in Figure 8 (FACS) and Figures 9 and 10 (confocal microscope). The results of this experiment are summarized in FIG. 11. This antibody internalization study using primary CLL cells and B cells, performed by flow cytometry and confocal microscopy, showed that the distribution of CHIR-12.12 uniformly on the cell surface even after 3 hours in binding to CD40 at 37 ° C. Shows that it is. In contrast, after binding at 37 ° C., rituximab was redistributed or internalized into the cap. These data indicate that the potent ADCC activity of CHIR-12.12 may be related to the ability to be uniformly expressed on the surface of target cells while allowing optimal interaction of effector cells. These results indicate that CHIR-12.12 is effective for potent mediation of ADCC on CLL cells in vivo.

Example  6: Rituxan  And CHIR By -12.12 Fc γ RIIIa  Combined Biacore  analysis

The affinity of the FcγRIIIa aa158F and aa158V alleles for CHIR-12.12 and Rituximab was compared by standard Biacore® analysis. CHIR-12.12 binds the aa158F allele with a 4.6-fold higher affinity than rituximab (2.8 μM vs. 13 μM KQ, respectively). The results of these experiments are summarized in the following table:

KD ( nM ) CHIR-12.12 Rituximab FcγRIIIa 158V  492  466 FcγRIIIa 158V 2800 13000

Example  7: NK  By working cells ADCC For Fc γ RIIIa  Polymorphic effect

Antibody-dependent cellular cytotoxicity (ADCC) is the major mechanism of activity of many commercial and test monoclonal antibodies. Rituximab, marketed as a therapeutic for vesicle non-Hodgkin's lymphoma (NHL) and active against other B-cell malignancies, is known to have ADCC as one of their primary active mechanisms. Notably, the clinical activity of Rituximab in NHL is associated with the FcγRIIIa genotype. Patients with FcγRIIIa 158aa polymorphism of V / V or V / F respond more to rituximab than patients who are F / F (eg, Cartron et al. Blood (2002), 99 (3): 754-). 758 or Dall'Ozzo et al. Cancer Res. (2004) 64: 4664-4669).

In these experiments, NK effector cells purified from multiple human donors expressing various FcγRIIIa aa158 polymorphisms were evaluated using human lymphoma Daudi cell lines as target cells (FIGS. 12 and 13). As shown in this figure, CHIR-12.12 induces potent ADCC by NK cells of all three genotypes. CHIR-12.12 ED _50S for lysis of Daudi cell lines is 4, 2, and 0.4 pM for F / F, V / F and V / V, respectively (FIG. 13). Rituximab ED _50S for lysis of the Daudi cell line is 53, 21, and 9 pM for F / F, V / F, and V / V, respectively (FIG. 13).

Purified NK effector cells from a number of human donors expressing various FcγRIIIa aa158 polymorphisms were evaluated using CLL cell lines as target cells (FIG. 14). CHIR-12.12 was found to be more potent mediator of ADCC than rituximab for all CLL patient cells tested (FIG. 14). These data mean that CHIR-12.12 is a more potent ADCC inhibitor than rituximab even with NK cells of aa158 V / F or F / F genotype.

This finding is surprising because CHIR-12.12 was expected to be significantly less potent in ADCC assays using NK cells with F / F or V / F's FcγRIIIa 158aa polymorphism than V / V. In addition, the clinical activity of Rituximab in NHL has been shown to be associated with the FcγRIIIa genotype. Patients with FcγRIIIa 158aa polymorphism of V / V or V / F are more responsive to rituximab than patients who are F / F. Rituximab is also an IgG1 monoclonal antibody that binds to an antigen expressed on the surface of B cells, and therefore it has been expected that CHIR-12.12 exhibits the same preference for FcγRIIIa-158 V polymorphism. Instead, CHIR-12.12 was found to induce potent ADCC into NK cells of all three genotypes.

Many modifications and other embodiments of the invention described herein will be devised by those skilled in the art so that this invention will retain the advantages of the invention set forth in the foregoing specification and associated drawings. Accordingly, the invention is not limited to the particular embodiments, and variations and other embodiments are intended to be included within the scope of the appended claims. Although specific terminology is employed, it is to be understood that this is inclusive, descriptive and not for purposes of limitation.

All documents and patent applications cited herein are hereby incorporated by reference in their entirety to the same extent as indicating that each individual document or patent application is individually and clearly incorporated by reference.

Applicant or representative file number PP028162.0002 (319129) International application number PCT / US2006 Directive on Deposited Microorganisms or Other Biological Substances (PCT Rule 13 bis )   A. The following instructions relate to deposited microorganisms or other biological materials referred to in line 25 on page 18 of this specification.  B. Confirmation of Deposits Additional deposits have been identified on the Annex  Depository American Type Culture Collection  Address of Depositary Virginia, USA 20110-2209, Manassas University Boulevard 10801  Deposited Date September 17, 2003  Approval number PTA-5543  C. Additional Information (if required) Continue this information on the separate sheet □   Page 48, line 13; P. 79, lines 20-23  D. Designated Country Directed  E. Attachment Instructions  The following instructions shall be submitted to the International Bureau.

Of microbial deposits for the purposes of patent procedures

For international approval Butafest  treaty

International Form

Deposits of organics submitted under Rule 7.3 and in accordance with Rule 10.2

Receipt of cases for submitted growth status

To: (name and address of trustee or agent)

Cairo Corporation

Recipient: Karen Van Nero

Emery Villa, California, USA 94608

Donor: Kyron Corporation

Accessory Identification Reference: Patent Deposit Name

Mouse hybridoma 131.2F8.5.9: CMCC # 12047 PTA-5542

Mouse hybridoma 153.8E2D10D6.12.12 CMCC # 12056 PTA-5543

This deposit entails the above-described scientific description 분류 proposed taxonomic description.

This deposit was received and approved by the International Depositary on 17 September 2003.

On request: X Provides information on claims of this strain for 30 years.

This strain may be used if the right to receive it is signed by the Patent Office under the Butafest Treaty, or if a US patent citing this strain has been issued and the publication of the strain has been instructed by the U.S. Patent and Trademark Office or Depositary. Could be.

If a culture dies or is damaged during the period of validity, the depositor is responsible for replacing it with the same live culture.

This strain will be maintained for at least thirty years from the date of deposit, or for the longer of five years after the most recent sample request. The United States and many other countries have signed the Butafest Treaty.

The viability of the cultures cited above was tested on September 23, 2003, by which time the cultures were alive.

International Depository: American Type Culture Collection, Virginia, USA 20110-2209 Manassas

Signature date: 2003. 10. 2

                                SEQUENCE LISTING <110> Aukerman, Sharon Lea       Luqman, Mohammad <120> Uses of Anti-CD40 Antibodies    <130> PP028162.0002 (319129) <150> 60 / 732,730 <151> 2005-11-01 <160> 9 FastSEQ for Windows Version 4.0 <210> 1 <211> 720 <212> DNA <213> Artificial Sequence <220> <223> Coding sequence for light chain of CHIR-12.12       human anti-CD40 antibody        <221> CDS <222> (1) ... (720) <400> 1 atg gcg ctc cct gct cag ctc ctg ggg ctg cta atg ctc tgg gtc tct 48 Met Ala Leu Pro Ala Gln Leu Leu Gly Leu Leu Met Leu Trp Val Ser  1 5 10 15   gga tcc agt ggg gat att gtg atg act cag tct cca ctc tcc ctg acc 96 Gly Ser Ser Gly Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Thr              20 25 30   gtc acc cct gga gag ccg gcc tcc atc tcc tgc agg tcc agt cag agc 144 Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser          35 40 45   ctc ctg tat agt aat gga tac aac tat ttg gat tgg tac ctg cag aag 192 Leu Leu Tyr Ser Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys      50 55 60   cca ggg cag tct cca cag gtc ctg atc tct ttg ggt tct aat cgg gcc 240 Pro Gly Gln Ser Pro Gln Val Leu Ile Ser Leu Gly Ser Asn Arg Ala  65 70 75 80   tcc ggg gtc cct gac agg ttc agt ggc agt gga tca ggc aca gat ttt 288 Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe                  85 90 95   aca ctg aaa atc agc aga gtg gag gct gag gat gtt ggg gtt tat tac 336 Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr             100 105 110   tgc atg caa gct cga caa act cca ttc act ttc ggc cct ggg acc aaa 384 Cys Met Gln Ala Arg Gln Thr Pro Phe Thr Phe Gly Pro Gly Thr Lys         115 120 125   gtg gat atc aga cga act gtg gct gca cca tct gtc ttc atc ttc ccg 432 Val Asp Ile Arg Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro     130 135 140   cca tct gat gag cag ttg aaa tct gga act gcc tct gtt gtg tgc ctg 480 Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu 145 150 155 160   ctg aat aac ttc tat ccc aga gag gcc aaa gta cag tgg aag gtg gat 528 Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp                 165 170 175   aac gcc ctc caa tcg ggt aac tcc cag gag agt gtc aca gag cag gac 576 Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp             180 185 190   agc aag gac agc acc tac agc ctc agc agc acc ctg acg ctg agc aaa 624 Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys         195 200 205   gca gac tac gag aaa cac aaa gtc tac gcc tgc gaa gtc acc cat cag 672 Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln     210 215 220   ggc ctg agc tcg ccc gtc aca aag agc ttc aac agg gga gag tgt tag 720 Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys * 225 230 235   <210> 2 <211> 239 <212> PRT <213> Artificial Sequence <220> <223> Light chain of CHIR-12.12 human anti-CD40 antibody <400> 2 Met Ala Leu Pro Ala Gln Leu Leu Gly Leu Leu Met Leu Trp Val Ser  1 5 10 15 Gly Ser Ser Gly Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Thr             20 25 30 Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser         35 40 45 Leu Leu Tyr Ser Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys     50 55 60 Pro Gly Gln Ser Pro Gln Val Leu Ile Ser Leu Gly Ser Asn Arg Ala 65 70 75 80 Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe                 85 90 95 Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr             100 105 110 Cys Met Gln Ala Arg Gln Thr Pro Phe Thr Phe Gly Pro Gly Thr Lys         115 120 125 Val Asp Ile Arg Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro     130 135 140 Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu 145 150 155 160 Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp                 165 170 175 Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp             180 185 190 Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys         195 200 205 Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln     210 215 220 Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 <210> 3 <211> 2016 <212> DNA <213> Artificial Sequence <220> <223> Coding sequence for heavy chain of CHIR-12.12       human anti-CD40 antibody (with introns)        <400> 3 atggagtttg ggctgagctg ggttttcctt gttgctattt taagaggtgt ccagtgtcag 60 gtgcagttgg tggagtctgg gggaggcgtg gtccagcctg ggaggtccct gagactctcc 120 tgtgcagcct ctggattcac cttcagtagc tatggcatgc actgggtccg ccaggctcca 180 ggcaaggggc tggagtgggt ggcagttata tcatatgagg aaagtaatag ataccatgca 240 gactccgtga agggccgatt caccatctcc agagacaatt ccaagatcac gctgtatctg 300 caaatgaaca gcctcagaac tgaggacacg gctgtgtatt actgtgcgag agatgggggt 360 atagcagcac ctgggcctga ctactggggc cagggaaccc tggtcaccgt ctcctcagca 420 agtaccaagg gcccatccgt cttccccctg gcgcccgcta gcaagagcac ctctgggggc 480 acagcggccc tgggctgcct ggtcaaggac tacttccccg aaccggtgac ggtgtcgtgg 540 aactcaggcg ccctgaccag cggcgtgcac accttcccgg ctgtcctaca gtcctcagga 600 ctctactccc tcagcagcgt ggtgaccgtg ccctccagca gcttgggcac ccagacctac 660 atctgcaacg tgaatcacaa gcccagcaac accaaggtgg acaagagagt tggtgagagg 720 ccagcacagg gagggagggt gtctgctgga agccaggctc agcgctcctg cctggacgca 780 tcccggctat gcagtcccag tccagggcag caaggcaggc cccgtctgcc tcttcacccg 840 gaggcctctg cccgccccac tcatgctcag ggagagggtc ttctggcttt ttccccaggc 900 tctgggcagg cacaggctag gtgcccctaa cccaggccct gcacacaaag gggcaggtgc 960 tgggctcaga cctgccaaga gccatatccg ggaggaccct gcccctgacc taagcccacc 1020 ccaaaggcca aactctccac tccctcagct cggacacctt ctctcctccc agattccagt 1080 aactcccaat cttctctctg cagagcccaa atcttgtgac aaaactcaca catgcccacc 1140 gtgcccaggt aagccagccc aggcctcgcc ctccagctca aggcgggaca ggtgccctag 1200 agtagcctgc atccagggac aggccccagc cgggtgctga cacgtccacc tccatctctt 1260 cctcagcacc tgaactcctg gggggaccgt cagtcttcct cttcccccca aaacccaagg 1320 acaccctcat gatctcccgg acccctgagg tcacatgcgt ggtggtggac gtgagccacg 1380 aagaccctga ggtcaagttc aactggtacg tggacggcgt ggaggtgcat aatgccaaga 1440 caaagccgcg ggaggagcag tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc 1500 tgcaccagga ctggctgaat ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc 1560 cagcccccat cgagaaaacc atctccaaag ccaaaggtgg gacccgtggg gtgcgagggc 1620 cacatggaca gaggccggct cggcccaccc tctgccctga gagtgaccgc tgtaccaacc 1680 tctgtcccta cagggcagcc ccgagaacca caggtgtaca ccctgccccc atcccgggag 1740 gagatgacca agaaccaggt cagcctgacc tgcctggtca aaggcttcta tcccagcgac 1800 atcgccgtgg agtgggagag caatgggcag ccggagaaca actacaagac cacgcctccc 1860 gtgctggact ccgacggctc cttcttcctc tatagcaagc tcaccgtgga caagagcagg 1920 tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac 1980 acgcagaaga gcctctccct gtctccgggt aaatga 2016 <210> 4 <211> 469 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain of CHIR-12.12 human anti-CD40 antibody <400> 4 Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Ile Leu Arg Gly  1 5 10 15 Val Gln Cys Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln             20 25 30 Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe         35 40 45 Ser Ser Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu     50 55 60 Glu Trp Val Ala Val Ile Ser Tyr Glu Glu Ser Asn Arg Tyr His Ala 65 70 75 80 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Ile                 85 90 95 Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Thr Glu Asp Thr Ala Val             100 105 110 Tyr Tyr Cys Ala Arg Asp Gly Gly Ile Ala Ala Pro Gly Pro Asp Tyr         115 120 125 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly     130 135 140 Pro Ser Val Phe Pro Leu Ala Pro Ala Ser Lys Ser Thr Ser Gly Gly 145 150 155 160 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val                 165 170 175 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe             180 185 190 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val         195 200 205 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val     210 215 220 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys 225 230 235 240 Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu                 245 250 255 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr             260 265 270 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Asp Val         275 280 285 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val     290 295 300 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 305 310 315 320 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu                 325 330 335 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala             340 345 350 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro         355 360 365 Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln     370 375 380 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 385 390 395 400 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr                 405 410 415 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu             420 425 430 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser         435 440 445 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser     450 455 460 Leu Ser Pro Gly Lys 465 <210> 5 <211> 469 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain of variant of CHIR-12.12 human       anti-CD40 antibody        <400> 5 Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Ile Leu Arg Gly  1 5 10 15 Val Gln Cys Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln             20 25 30 Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe         35 40 45 Ser Ser Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu     50 55 60 Glu Trp Val Ala Val Ile Ser Tyr Glu Glu Ser Asn Arg Tyr His Ala 65 70 75 80 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Ile                 85 90 95 Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Thr Glu Asp Thr Ala Val             100 105 110 Tyr Tyr Cys Ala Arg Asp Gly Gly Ile Ala Ala Pro Gly Pro Asp Tyr         115 120 125 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly     130 135 140 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 145 150 155 160 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val                 165 170 175 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe             180 185 190 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val         195 200 205 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val     210 215 220 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys 225 230 235 240 Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu                 245 250 255 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr             260 265 270 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Asp Val         275 280 285 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val     290 295 300 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 305 310 315 320 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu                 325 330 335 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala             340 345 350 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro         355 360 365 Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln     370 375 380 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 385 390 395 400 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr                 405 410 415 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu             420 425 430 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser         435 440 445 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser     450 455 460 Leu Ser Pro Gly Lys 465 <210> 6 <211> 612 <212> DNA <213> Homo sapiens <220> <221> misc_feature <222> (0) ... (0) <223> Coding sequence for short isoform of human CD40 <221> CDS <222> (1) ... (612) <400> 6 atg gtt cgt ctg cct ctg cag tgc gtc ctc tgg ggc tgc ttg ctg acc 48 Met Val Arg Leu Pro Leu Gln Cys Val Leu Trp Gly Cys Leu Leu Thr  1 5 10 15   gct gtc cat cca gaa cca ccc act gca tgc aga gaa aaa cag tac cta 96 Ala Val His Pro Glu Pro Pro Thr Ala Cys Arg Glu Lys Gln Tyr Leu              20 25 30   ata aac agt cag tgc tgt tct ttg tgc cag cca gga cag aaa ctg gtg 144 Ile Asn Ser Gln Cys Cys Ser Leu Cys Gln Pro Gly Gln Lys Leu Val          35 40 45   agt gac tgc aca gag ttc act gaa acg gaa tgc ctt cct tgc ggt gaa 192 Ser Asp Cys Thr Glu Phe Thr Glu Thr Glu Cys Leu Pro Cys Gly Glu      50 55 60   agc gaa ttc cta gac acc tgg aac aga gag aca cac tgc cac cag cac 240 Ser Glu Phe Leu Asp Thr Trp Asn Arg Glu Thr His Cys His Gln His  65 70 75 80   aaa tac tgc gac ccc aac cta ggg ctt cgg gtc cag cag aag ggc acc 288 Lys Tyr Cys Asp Pro Asn Leu Gly Leu Arg Val Gln Gln Lys Gly Thr                  85 90 95   tca gaa aca gac acc atc tgc acc tgt gaa gaa ggc tgg cac tgt acg 336 Ser Glu Thr Asp Thr Ile Cys Thr Cys Glu Glu Gly Trp His Cys Thr             100 105 110   agt gag gcc tgt gag agc tgt gtc ctg cac cgc tca tgc tcg ccc ggc 384 Ser Glu Ala Cys Glu Ser Cys Val Leu His Arg Ser Cys Ser Pro Gly         115 120 125   ttt ggg gtc aag cag att gct aca ggg gtt tct gat acc atc tgc gag 432 Phe Gly Val Lys Gln Ile Ala Thr Gly Val Ser Asp Thr Ile Cys Glu     130 135 140   ccc tgc cca gtc ggc ttc ttc tcc aat gtg tca tct gct ttc gaa aaa 480 Pro Cys Pro Val Gly Phe Phe Ser Asn Val Ser Ser Ala Phe Glu Lys 145 150 155 160   tgt cac cct tgg aca agg tcc cca gga tcg gct gag agc cct ggt ggt 528 Cys His Pro Trp Thr Arg Ser Pro Gly Ser Ala Glu Ser Pro Gly Gly                 165 170 175   gat ccc cat cat ctt cgg gat cct gtt tgc cat cct ctt ggt gct ggt 576 Asp Pro His His Leu Arg Asp Pro Val Cys His Pro Leu Gly Ala Gly             180 185 190   ctt tat caa aaa ggt ggc caa gaa gcc aac caa taa 612 Leu Tyr Gln Lys Gly Gly Gln Glu Ala Asn Gln *         195 200   <210> 7 <211> 203 <212> PRT <213> Homo sapiens <400> 7 Met Val Arg Leu Pro Leu Gln Cys Val Leu Trp Gly Cys Leu Leu Thr  1 5 10 15 Ala Val His Pro Glu Pro Pro Thr Ala Cys Arg Glu Lys Gln Tyr Leu             20 25 30 Ile Asn Ser Gln Cys Cys Ser Leu Cys Gln Pro Gly Gln Lys Leu Val         35 40 45 Ser Asp Cys Thr Glu Phe Thr Glu Thr Glu Cys Leu Pro Cys Gly Glu     50 55 60 Ser Glu Phe Leu Asp Thr Trp Asn Arg Glu Thr His Cys His Gln His 65 70 75 80 Lys Tyr Cys Asp Pro Asn Leu Gly Leu Arg Val Gln Gln Lys Gly Thr                 85 90 95 Ser Glu Thr Asp Thr Ile Cys Thr Cys Glu Glu Gly Trp His Cys Thr             100 105 110 Ser Glu Ala Cys Glu Ser Cys Val Leu His Arg Ser Cys Ser Pro Gly         115 120 125 Phe Gly Val Lys Gln Ile Ala Thr Gly Val Ser Asp Thr Ile Cys Glu     130 135 140 Pro Cys Pro Val Gly Phe Phe Ser Asn Val Ser Ser Ala Phe Glu Lys 145 150 155 160 Cys His Pro Trp Thr Arg Ser Pro Gly Ser Ala Glu Ser Pro Gly Gly                 165 170 175 Asp Pro His His Leu Arg Asp Pro Val Cys His Pro Leu Gly Ala Gly             180 185 190 Leu Tyr Gln Lys Gly Gly Gln Glu Ala Asn Gln         195 200 <210> 8 <211> 834 <212> DNA <213> Homo sapiens <220> <221> misc_feature <222> (0) ... (0) <223> Coding sequence for long isoform of human CD40 <221> CDS <222> (1) ... (834) <400> 8 atg gtt cgt ctg cct ctg cag tgc gtc ctc tgg ggc tgc ttg ctg acc 48 Met Val Arg Leu Pro Leu Gln Cys Val Leu Trp Gly Cys Leu Leu Thr  1 5 10 15   gct gtc cat cca gaa cca ccc act gca tgc aga gaa aaa cag tac cta 96 Ala Val His Pro Glu Pro Pro Thr Ala Cys Arg Glu Lys Gln Tyr Leu              20 25 30   ata aac agt cag tgc tgt tct ttg tgc cag cca gga cag aaa ctg gtg 144 Ile Asn Ser Gln Cys Cys Ser Leu Cys Gln Pro Gly Gln Lys Leu Val          35 40 45   agt gac tgc aca gag ttc act gaa acg gaa tgc ctt cct tgc ggt gaa 192 Ser Asp Cys Thr Glu Phe Thr Glu Thr Glu Cys Leu Pro Cys Gly Glu      50 55 60   agc gaa ttc cta gac acc tgg aac aga gag aca cac tgc cac cag cac 240 Ser Glu Phe Leu Asp Thr Trp Asn Arg Glu Thr His Cys His Gln His  65 70 75 80   aaa tac tgc gac ccc aac cta ggg ctt cgg gtc cag cag aag ggc acc 288 Lys Tyr Cys Asp Pro Asn Leu Gly Leu Arg Val Gln Gln Lys Gly Thr                  85 90 95   tca gaa aca gac acc atc tgc acc tgt gaa gaa ggc tgg cac tgt acg 336 Ser Glu Thr Asp Thr Ile Cys Thr Cys Glu Glu Gly Trp His Cys Thr             100 105 110   agt gag gcc tgt gag agc tgt gtc ctg cac cgc tca tgc tcg ccc ggc 384 Ser Glu Ala Cys Glu Ser Cys Val Leu His Arg Ser Cys Ser Pro Gly         115 120 125   ttt ggg gtc aag cag att gct aca ggg gtt tct gat acc atc tgc gag 432 Phe Gly Val Lys Gln Ile Ala Thr Gly Val Ser Asp Thr Ile Cys Glu     130 135 140   ccc tgc cca gtc ggc ttc ttc tcc aat gtg tca tct gct ttc gaa aaa 480 Pro Cys Pro Val Gly Phe Phe Ser Asn Val Ser Ser Ala Phe Glu Lys 145 150 155 160   tgt cac cct tgg aca agc tgt gag acc aaa gac ctg gtt gtg caa cag 528 Cys His Pro Trp Thr Ser Cys Glu Thr Lys Asp Leu Val Val Gln Gln                 165 170 175   gca ggc aca aac aag act gat gtt gtc tgt ggt ccc cag gat cgg ctg 576 Ala Gly Thr Asn Lys Thr Asp Val Val Cys Gly Pro Gln Asp Arg Leu             180 185 190   aga gcc ctg gtg gtg atc ccc atc atc ttc ggg atc ctg ttt gcc atc 624 Arg Ala Leu Val Val Ile Pro Ile Ile Phe Gly Ile Leu Phe Ala Ile         195 200 205   ctc ttg gtg ctg gtc ttt atc aaa aag gtg gcc aag aag cca acc aat 672 Leu Leu Val Leu Val Phe Ile Lys Lys Val Ala Lys Lys Pro Thr Asn     210 215 220   aag gcc ccc cac ccc aag cag gaa ccc cag gag atc aat ttt ccc gac 720 Lys Ala Pro His Pro Lys Gln Glu Pro Gln Glu Ile Asn Phe Pro Asp 225 230 235 240   gat ctt cct ggc tcc aac act gct gct cca gtg cag gag act tta cat 768 Asp Leu Pro Gly Ser Asn Thr Ala Ala Pro Val Gln Glu Thr Leu His                 245 250 255   gga tgc caa ccg gtc acc cag gag gat ggc aaa gag agt cgc atc tca 816 Gly Cys Gln Pro Val Thr Gln Glu Asp Gly Lys Glu Ser Arg Ile Ser             260 265 270   gtg cag gag aga cag tga 834 Val Gln Glu Arg Gln *         275   <210> 9 <211> 277 <212> PRT <213> Homo sapiens <400> 9 Met Val Arg Leu Pro Leu Gln Cys Val Leu Trp Gly Cys Leu Leu Thr  1 5 10 15 Ala Val His Pro Glu Pro Pro Thr Ala Cys Arg Glu Lys Gln Tyr Leu             20 25 30 Ile Asn Ser Gln Cys Cys Ser Leu Cys Gln Pro Gly Gln Lys Leu Val         35 40 45 Ser Asp Cys Thr Glu Phe Thr Glu Thr Glu Cys Leu Pro Cys Gly Glu     50 55 60 Ser Glu Phe Leu Asp Thr Trp Asn Arg Glu Thr His Cys His Gln His 65 70 75 80 Lys Tyr Cys Asp Pro Asn Leu Gly Leu Arg Val Gln Gln Lys Gly Thr                 85 90 95 Ser Glu Thr Asp Thr Ile Cys Thr Cys Glu Glu Gly Trp His Cys Thr             100 105 110 Ser Glu Ala Cys Glu Ser Cys Val Leu His Arg Ser Cys Ser Pro Gly         115 120 125 Phe Gly Val Lys Gln Ile Ala Thr Gly Val Ser Asp Thr Ile Cys Glu     130 135 140 Pro Cys Pro Val Gly Phe Phe Ser Asn Val Ser Ser Ala Phe Glu Lys 145 150 155 160 Cys His Pro Trp Thr Ser Cys Glu Thr Lys Asp Leu Val Val Gln Gln                 165 170 175 Ala Gly Thr Asn Lys Thr Asp Val Val Cys Gly Pro Gln Asp Arg Leu             180 185 190 Arg Ala Leu Val Val Ile Pro Ile Ile Phe Gly Ile Leu Phe Ala Ile         195 200 205 Leu Leu Val Leu Val Phe Ile Lys Lys Val Ala Lys Lys Pro Thr Asn     210 215 220 Lys Ala Pro His Pro Lys Gln Glu Pro Gln Glu Ile Asn Phe Pro Asp 225 230 235 240 Asp Leu Pro Gly Ser Asn Thr Ala Ala Pro Val Gln Glu Thr Leu His                 245 250 255 Gly Cys Gln Pro Val Thr Gln Glu Asp Gly Lys Glu Ser Arg Ile Ser             260 265 270 Val Gln Glu Arg Gln         275  

Claims (60)

A method of treating cancer or precancerous conditions associated with CD40-expressing cells of a human patient, wherein the human patient is heterozygous or homozygous for FcγRIIIa-158F (genotype V / F or F / F) A method comprising administering a therapeutically or prophylactically effective amount of an anti-CD40 antibody. The method of claim 1, wherein the cancer or precancerous condition is a B-cell family cancer. The method of claim 2, wherein the B-type cancer is acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), prelymphocytic leukemia (PLL), small lymphocytic leukemia (SLL), hair cells Leukemia, Hodgkin's Disease, Multiple Myeloma, Waldenstrom's Macroglobulinemia, Heavy Chain Diseases, and Lymphomas, such as extensive bovine lymphoma, vesicles, DLBCL, mucosal associated lymphoid tissue lymphoma, monocyte B cell lymphoma, spleen lymphoma, lymphoma Granulomatosis, endovascular lymphoma, immune blast lymphoma, and AIDS-associated lymphoma. The method of claim 1, wherein the cancer or precancerous condition is a non-B cell hematologic malignancy. The method of claim 4, wherein the non-B cell hematologic malignancy is acute myeloid leukemia. The method of claim 1, wherein the cancer or precancerous condition is a solid tumor. The method of claim 6, wherein the solid tumor is ovarian cancer, lung cancer (eg, non-small cell lung cancer of squamous cell carcinoma, adenocarcinoma, and giant cell carcinoma type, and small cell lung cancer), breast cancer, colon cancer, kidney cancer (eg, Renal cell carcinoma), bladder cancer, liver cancer (including hepatocellular carcinoma), gastric cancer, cervical cancer, prostate cancer, nasal pharyngeal cancer, and thyroid cancer (eg thyroid papillary carcinoma), skin cancers such as melanoma And sarcoma, including, for example, osteosarcoma and Ewing sarcoma. The method of claim 1, wherein the cancer or precancerous condition is a cancer or precancerous condition associated with CD20-expressing cells. The method of claim 8, wherein the cancer or precancerous condition is a B cell malignancy. The method of claim 8, wherein the cancer or precancerous condition is a cancer or precancerous condition associated with cells expressing both CD40 and CD20. The method of claim 10, wherein the cancer or precancerous condition is a B cell malignancy. 12. The method of any one of claims 1-11, wherein the anti-CD40 antibody is administered by parenteral route of administration. The method of claim 12, wherein the anti-CD40 antibody is administered intravenously or subcutaneously. Therapeutic treatment of anti-CD40 antibodies in the manufacture of a medicament for the treatment of cancer or precancerous conditions associated with CD40-expressing cells in human patients who are heterozygous or homozygous (genotype V / F or F / F) to FcγRIIIa-158F Or the use of a prophylactically effective amount. Use according to claim 14, wherein the cancer or precancerous condition is a B-cell family cancer. The method of claim 15, wherein the cancer of the B-cell family is acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), prelymphocytic leukemia (PLL), bovine lymphocytic leukemia (SLL), hair cells Leukemia, Hodgkin's Disease, Multiple Myeloma, Waldenstrom's Macroglobulinemia, Heavy Chain Diseases, and Lymphomas, such as extensive bovine lymphoma, vesicles, DLBCL, mucosal associated lymphoid tissue lymphoma, monocyte B cell lymphoma, spleen lymphoma, lymphoma Use selected from the group consisting of granulomatosis, endovascular lymphoma, immune blast lymphoma, and AIDS-associated lymphoma. Use according to claim 14, wherein the cancer or precancerous condition is a non-B cell hematologic malignancy. 18. The use of claim 17, wherein the non-B cell hematologic malignancy is acute myeloid leukemia. Use according to claim 14, wherein the cancer or precancerous condition is a solid tumor. The method of claim 19, wherein the solid tumor is ovarian cancer, lung cancer (eg, non-small cell lung cancer of squamous cell carcinoma, adenocarcinoma, and giant cell carcinoma type, and small cell lung cancer), breast cancer, colon cancer, kidney cancer (eg, Renal cell carcinoma), bladder cancer, liver cancer (including hepatocellular carcinoma), gastric cancer, cervical cancer, prostate cancer, nasal pharyngeal cancer, and thyroid cancer (eg thyroid papillary carcinoma), skin cancers such as melanoma And, for example, sarcoma, including osteosarcoma and Ewing sarcoma. 15. The use of claim 14, wherein the cancer or precancerous condition is a cancer or precancerous condition associated with CD20-expressing cells. Use according to claim 21, wherein the cancer or precancerous condition is a B cell malignancy. Use according to claim 21, wherein the cancer or precancerous condition is a cancer or precancerous condition associated with cells expressing both CD40 and CD20. Use according to claim 23, wherein the cancer or precancerous condition is a B cell malignancy. Use according to any one of claims 14 to 24, characterized in that the medicament is formulated for administration by the parenteral route of administration. The use of claim 25, wherein the medicament is formulated for intravenous or subcutaneous administration. Inhibit antibody production by B cells in human patients that are heterozygous or homozygous (genotype V / F or F / F) to FcγRIIIa-158F, comprising administering an effective amount of an anti-CD40 antibody to the human patient How to. The method of claim 27, wherein the human patient has a cancer or precancerous condition associated with CD40-expressing cells. Use of an effective amount of an anti-CD40 antibody in the manufacture of a medicament for the inhibition of antibody production by B cells in human patients who are heterozygous or homozygous (genotype V / F or F / F) to FcγRIIIa-158F. The method or use according to any one of the preceding claims, wherein said anti-CD40 antibody is a human monoclonal antibody. The method or use of claim 30, wherein the human anti-CD40 monoclonal antibody comprises a human IgG1 heavy chain constant region. The method or use of claim 31, wherein the human IgG1 heavy chain constant region comprises the amino acid sequence recited in SEQ ID NO: 4 or SEQ ID NO: 5. The method or use of any one of the preceding claims, wherein said anti-CD40 antibody lacks significant agonist activity. The method or use according to any one of the preceding claims, wherein said anti-CD40 antibody is an antagonist of CD40-CD40L signal on CD40-expressing cells. The method of claim 1, wherein the anti-CD40 antibody is: a) monoclonal antibody CHIR-12.12; b) monoclonal antibodies produced by hybridoma cell line 12.12; c) the sequence shown in SEQ ID NO: 2, the sequence shown in SEQ ID NO: 4, the sequence shown in SEQ ID NO: 5, both sequences shown in SEQ ID NO: 2 and SEQ ID NO: 4, and SEQ ID NO Monoclonal antibodies comprising an amino acid sequence selected from the group consisting of: 2 and both sequences shown in SEQ ID NO: 5; d) a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of a sequence shown in SEQ ID NO: 1, a sequence shown in SEQ ID NO: 3, and both sequences shown in SEQ ID NO: 1 and SEQ ID NO: 3 Monoclonal antibodies having amino acid sequences encoded by; e) monoclonal antibodies that bind to epitopes bindable to monoclonal antibodies produced by the hybridoma cell line 12.12; f) a monoclonal antibody that binds to an epitope comprising residues 82-87 of the human CD40 sequence shown in SEQ ID NO: 7 or SEQ ID NO: 9; g) a monoclonal antibody that binds to an epitope comprising residues 82-89 of the human CD40 sequence shown in SEQ ID NO: 7 or SEQ ID NO: 9; h) monoclonal antibodies that compete with monoclonal antibody CHIR-12.12 in a competitive binding assay; i) the monoclonal antibody of item a) above or the monoclonal antibody of any one of items c) -h) above, which is recombinantly produced; And j) a monoclonal antibody which is an antigen-binding fragment of the monoclonal antibody of any one of the preceding items a) -i), wherein said fragment has the ability to specifically bind a human CD40 antigen Method or use, characterized in that selected from the group consisting of. The method or use of claim 35, wherein the anti-CD40 antibody is a monoclonal antibody CHIR-12.12. 36. The method or use of claim 35, wherein the antigen-binding fragment is selected from the group consisting of Fab fragments, F (ab ') 2 fragments, Fv fragments, and single-chain Fv fragments. Methods of identifying human patients with cancer or precancerous conditions treatable with anti-CD40 antibodies resistant to the treatment of Rituximab (Rituxan®): a) identifying a human patient with a cancer or precancerous condition associated with CD40-expressing cells; And b) determining the FcγRIIIa-158 genotype (V / V, V / F or F / F) of the human patient; Wherein if the human patient is heterozygous or homozygous for FcγRIIIa-158F (genotype V / F or F / F), the cancer or precancerous condition is treatable with an anti-CD40 antibody. As a method of selecting antibody therapies for the treatment of human patients with cancer or precancerous conditions resistant to the treatment of Rituximab (Rituxan®): a) identifying a human patient that is resistant to the treatment of Rituximab (Rituxan®) and has a cancer or precancerous condition associated with CD40-expressing cells; And b) determining the FcγRIIIa-158 genotype (V / V, V / F or F / F) of the human patient; Wherein the human patient is heterozygous or homozygous for FcγRIIIa-158F (genotype V / F or F / F), wherein the anti-CD40 antibody is selected for treatment of the cancer or precancerous condition . The method or use according to any one of the preceding claims, wherein said human patient is resistant to anticancer drug therapy. The method of claim 40, wherein the human patient is resistant to anti-CD20 monoclonal antibody therapy. The method of claim 41, wherein the human patient is resistant to anti-CD20 monoclonal antibody therapy. 42. The method of claim 41, wherein the human patient is nonresponsive to anti-CD20 monoclonal antibody therapy. 44. The method of any one of claims 41-43, wherein the anti-CD20 monoclonal antibody is Rituximab (Rituxin®). A kit for identifying human patients with cancer or precancerous conditions treatable with an anti-CD40 antibody, comprising a reagent for determining the FcγRIIIa-158 genotype of a human patient. A kit for selecting antibody therapy for the treatment of human patients with cancer or precancerous conditions associated with CD40-expressing cells, comprising reagents for determining the FcγRIIIa-158 genotype of a human patient. 47. A microarray according to claim 45 or 46 comprising at least 10 nucleotides in length and comprising at least one probe having a sequence suitable for determining the FcγRIIIa-158 genotype of a human patient. Kit characterized by the above. 47. The kit of claim 45 or 46, comprising an oligonucleotide suitable for use as a primer in polymerase-catalytic amplification of the genomic region encoding amino acid 158 of Fc [gamma] RIIIa. 47. The kit of claim 45 or 46, comprising one or more restriction enzymes suitable for determining FcγRIIIa-158 genotyping in human patients. A method of treating a cancer or precancerous condition associated with a CD40-expressing cell of a human patient, the method comprising administering to the human patient a therapeutically or prophylactically effective amount of an anti-CD40 antibody, wherein, after administration, an anti-CD40 antibody Is not significantly internalized by CD40-expressing cells. A method of treating a cancer or precancerous condition associated with a CD40-expressing cell of a human patient, the method comprising administering to said human patient a therapeutically or prophylactically effective amount of an anti-CD40 antibody, wherein, after administration, an anti-CD40 antibody Is distributed substantially uniformly on the surface of CD40-expressing cells. A method of treating a cancer or precancerous condition associated with a CD40-expressing cell of a human patient, comprising administering an anti-CD40 antibody to the human patient, wherein, after administration, a therapeutically or prophylactically effective amount of an anti-CD40 antibody Which is presented on the surface of CD40-expressing cells in said human patient. 53. The antibody-dependent cell cell of any one of claims 1-37 or 50-52, wherein said method or use is a CD40-expressing cell by FcγRIIIa-expressing natural killer (NK) cells in a human patient. A method or use characterized by causing toxicity (ADCC). The antibody of any one of claims 1-37 or 50-52, wherein the anti-CD40 antibody is more potent than Rituximab in the analysis of antibody-dependent cellular cytotoxicity (ADCC). Wherein the assay comprises culturing the CD40-expressing cells and the CD20-expressing cells with human NK cells isolated in the presence of the antibody of interest. The method of any one of claims 1-37 or 50-52, wherein the anti-CD40 antibody is more potent than Rituximab in a nude mouse xenograft tumor model. Or uses. 56. The method of claim 55, wherein said nude mouse xenograft tumor model uses a Daudi human lymphoma cell line of myeloma cell line. 53. The antibody of any one of claims 1-37 or 50-52, wherein the anti-CD40 antibody is human at an affinity (K _D ) of at least about 10 ^-6 M to at least about 10 ^-12 M. A method or use characterized by binding to CD40. The antibody of any one of claims 1-37 or 50-52, wherein the anti-CD40 antibody binds to human FcγRIIIa-158V with an affinity (K _D ) of at least about 0.5 μM. How or Use. The antibody of any one of claims 1-37 or 50-52, wherein the anti-CD40 antibody binds to human FcγRIIIa-158F with an affinity (K _D ) of at least about 12 μM. How or Use. The antibody of any one of claims 1-37 or 50-52, wherein the anti-CD40 antibody binds to human FcγRIIIa-158V with an affinity (K _D ) of at least about 0.5 μM and at least about A method or use characterized by binding to human FcγRIIIa-158F with an affinity (K _D ) of 12 μM.

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